Transglutaminase-catalyzed preparation of chitosan-ovalbumin films

Microbial transglutaminase was employed as catalyst for preparing chitosan–ovalbumin films. The films showed low solubility at a wide range of pH and underwent to a good enzymatic hydrolysis with trypsin. The degree of swelling was reduced and the mechanical resistance of the chitosan–ovalbumin films increased from 24 to 35MPa after enzymatic treatment with transglutaminase. The barrier efficiency toward water vapour was slightly improved for the films prepared by transglutaminase-mediated cross-linking

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

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

Food-dependent Cushing's syndrome: from molecular characterization to therapeutical results

ObjectiveCortisol secretion in ACTH-independent macronodular adrenal hyperplasia (AIMAH) may be regulated by the aberrant expression of several G-protein-coupled receptors. Bilateral adrenalectomy is the treatment of choice in most cases. We searched for aberrant receptor expression in a patient with AIMAH and evaluated the response to medical and surgical treatment.PatientA 35-year-old woman with amenorrhea, hirsutism, and hypertension presented ACTH-independent cortisol secretion with high androgen levels. Abdominal computed tomography showed bilateral adrenal macronodules (4.5 cm right and 1.0 cm left). Scintigraphy with I131-norcholesterol showed bilateral uptake, prevalent on the right side. Several in vivo stimulation tests were assessed before and after treatment and in vitro studies were performed after unilateral adrenalectomy.ResultsPlasma cortisol increased after a standard meal test (60%) and oral glucose loading (147%), and the response was blunted by pretreatment with 100 μg s.c. octreotide. The therapy with long-acting release octreotide (octreotide-LAR) showed an improvement in urinary free cortisol (UFC) levels. Unilateral adrenalectomy was performed and histopathology revealed macronodular AIMAH. Cortisol and androgens increased after perifusion of tumoral tissue with glucose-dependent insulinotropic polypeptide (GIP), and GIP and LH-receptor overexpression was found in both the adrenal nodules and the adjacent cortex. After surgery, UFC and androgen levels normalized followed by clinical improvement.ConclusionsGIP and LH-receptor expression may coexist in AIMAH, influencing the functional and morphological phenotype. Aberrant hormone receptor expression enables specific pharmacological treatment, but long-term studies are needed to evaluate its real efficacy. Unilateral adrenalectomy may be a safe initial option, particularly for asymmetric bilateral adrenal enlargements

Meta-GWAS Reveals Novel Genetic Variants Associated with Urinary Excretion of Uromodulin

Background Uromodulin, the most abundant protein excreted in normal urine, plays major roles in kidney physiology and disease. The mechanisms regulating the urinary excretion of uromodulin remain essentially unknown. Methods We conducted a meta-analysis of genome-wide association studies for raw (uUMOD) and indexed to creatinine (uUCR) urinary levels of uromodulin in 29,315 individuals of European ancestry from 13 cohorts. We tested the distribution of candidate genes in kidney segments and investigated the effects of keratin-40 (KRT40) on uromodulin processing. Results Two genome-wide significant signals were identified for uUMOD: a novel locus (P 1.24E-08) over the KRT40 gene coding for KRT40, a type 1 keratin expressed in the kidney, and the UMOD-PDILT locus (P 2.17E-88), with two independent sets of single nucleotide polymorphisms spread over UMOD and PDILT. Two genome-wide significant signals for uUCR were identified at the UMOD-PDILT locus and at the novel WDR72 locus previously associated with kidney function. The effect sizes for rs8067385, the index single nucleotide polymorphism in the KRT40 locus, were similar for both uUMOD and uUCR. KRT40 colocalized with uromodulin and modulating its expression in thick ascending limb (TAL) cells affected uromodulin processing and excretion. Conclusions Common variants in KRT40,WDR72, UMOD, and PDILT associate with the levels of uromodulin in urine. The expression of KRT40 affects uromodulin processing in TAL cells. These results, although limited by lack of replication, provide insights into the biology of uromodulin, the role of keratins in the kidney, and the influence of the UMOD-PDILT locus on kidney function

A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

BACKGROUND: TGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. METHODS: In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. RESULTS: In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity. CONCLUSIONS: A distal region of the TGM1 gene promoter, containing AP1 and Sp1 binding sites, is evolutionarily conserved and responsible for high level expression in transgenic mice and in transfected keratinocyte cultures

ICAR: endoscopic skull‐base surgery

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Effects of the axial force eccentricity on the time-variant structural reliability of aging r.c. cross-sections subjected to chloride-induced corrosion

Reinforced concrete structures are generally affected by degradation phenomena, which may include changes in strength and stiffness beyond the baseline conditions which are assumed in structural design. Some of these aging effects may cause component or system strengths to degrade over time, particularly when the concrete is exposed to an aggressive environment which may increase the risk of structural failure. For r.c. structures, due to the uncertainties in material and geometrical properties, in the magnitude and distribution of the loads, in the physical parameters which define the deterioration process, the structural safety should realistically be considered time-variant. In this context, this paper implements a computational probabilistic approach to predict the time-evolution of the mechanical and geometrical properties of a r.c. structural element (i.e., bridge pier) subjected to corrosion-induced deterioration as a consequence of the diffusive attack of chlorides in order to evaluate its service life. Adopting appropriate degradation models of the material properties, concrete and reinforcing steel, as well as assuming appropriate probability density functions related to mechanical and deterioration parameters, the proposed sectional approach is based on Monte Carlo simulations in order to evaluate time-variant axial force-bending moment resistance domains, with the aim to estimate the time-variant reliability index β for different axial force eccentricity values. Finally, an application of the proposed methodology to estimate the expected lifetime of a deteriorating r.c. bridge pier is described and discussed

Keratinocytes transglutaminase promoter analysis: identificafion of a functional response element

Keratinocyte transglutaminase catalyzes isopeptide bond formation to yield the highly insoluble crosslinked envelope during terminal differentiation of epidermal cells, Transcriptional response elements were identified in the 5'-flanking DNA of the gene for this enzyme by a combination of transient transfection and electrophoretic mobility shift analyses, Since human keratinocytes transcribed ineffectively transfected transglutaminase flanking DNA, a key feature of these experiments was the use of rat bladder epithelial cells as recipients, Serial deletion experiments identified by transient transfection an important response region containing three putative AP2-like response elements approximately 0.5 kilobases from the transcription initiation site, Oligonucleotides, each containing a single one of the elements, formed specific complexes with keratinocyte nuclear proteins, Two of the response elements were found to be functional by transfection in site-specific deletion experiments, Of these one formed specific DNA-protein complexes with nuclear proteins only from cells exhibiting keratinocyte differentiation. UV cross-linking experiments estimated the protein component of the complex to be approximate to 85 kDa, This response element alone increased substantially the transcription of a minimal transglutaminase promoter in transient transfections. Further characterization of the putative transcription factor binding to this response element may provide insight into the regulation of keratinocyte transglutaminase