pH-induced structural changes of tyrosinase from Agaricus bisporus using fluorescence and in silico methods.

BACKGROUND: Tyrosinases are involved in enzymatic browning reactions in damaged fruits during post-harvest handling and processing. The overall structure of tyrosinase from Agaricus bisporus mushrooms at different pH values was monitored using fluorescence spectroscopy and molecular dynamics simulations. RESULTS: When the pH value was increased from 6.0 to 9.0, the protein passed through several structural intermediates, including the tetramer, trimer and dimer stages. Changes in the secondary and tertiary structure of tyrosinase at neutral pH were outlined after running molecular dynamics simulations. A detailed check at the single-molecule level by means of molecular modeling tools suggested that the most important contribution to the fluorescence intensity is given by the H subunits with seven Trp and nine Tyr residues exposed to the solvent, whereas the lectin-like folded L subunits have only six Trp and three Tyr residues, of which only Trp(15) , Trp(59) and Trp(93) are partially exposed to the solvent. CONCLUSIONS: The results indicated that the enzyme was sensitive to pH. The experimental results revealed the unfolding of the native tetrameric enzyme in acidic pH range, causing exposure of the hydrophobic residues.

Influence of tumor topography on clinical-morphological profile of laryngeal malignancies.

AIM: Laryngeal cancers are redoubtable because they are still diagnosed in advanced stages which results in poor survival and the decline of life quality. The authors intend to identify if the tumor topography influences clinical behavior, the morphological profile and therapeutic strategy. PATIENTS, MATERIALS AND METHODS: The study group included 188 patients with laryngeal malignancies diagnosed and treated in an Ear, Nose and Throat (ENT) Department. The patients have been divided into four groups according to the tumor topography and extension. Three categories of parameters were defined (epidemiological, clinical, and morphological) and analyzed comparatively between the four groups using filter scales and the χ² (chi-squared) correlation test. RESULTS: Epidemiological parameters (sex, age, socio-economic status) showed no significant differences between the four groups. Clinical parameters (symptoms, lymphadenopathies, surgical procedures, and hospitalization) instead registered significant differences between the four groups. Morphological parameters (longitudinal diameter, transverse diameter, shape, gross aspect, histopathological aspect, grade, local invasion - pT, lymph node invasion - pN, metastases - pM and tumor stage), excepting shape, registered too significant differences between the four groups. The analysis of the whole set of parameters in each group revealed different, distinct profiles for each of the topographic groups, especially for glottic and large tumors. Our results concerning the entire series of tumors ranged in the limits of variation of each of the parameters observed in the literature. CONCLUSIONS: Our study revealed that tumors placed in different regions of the larynx have distinct profiles from epidemiological, clinical, and morphological points of view. However, the profile of our entire group of tumors proved to be comparable with the literature data.

Multicenter Epidemiological Study of Nosebleeds Requiring Hospitalization.

Epistaxis is a common presenting symptom either emergency condition or ambulatory in Otorhinolaryngology, affecting people of all ages. A multicentric retrospective descriptive study of 380 patients who were hospitalized with epistaxis over a 3.8 year period was carried out. Data collected was analyzed using IBM SPSS Statistics 25 and illustrated using Microsoft Office/Word 2016. The current study showed that male adults after 60 years old are most affected by nasal bleeding. More than half of hospitalized patients have severe epistaxis at the time of hospitalization. However, further studies are necessary to be done in order to fully elucidate the epidemiology of nosebleeds requiring hospitalization.

Etiological Profile of Epistaxis: Pre-Pandemic Versus Pandemic.

Nosebleed or epistaxis is one of the most common forms of presenting an emergency in the ENT field. Since March 11, 2020, the World Health Organisation has proclaimed COVID-19 a global pandemic, and the world has been closed down. The main objective of the study is to analyse and compare the dynamics of epistaxis aetiology among the cases that required hospitalisation in the pre-pandemic period and the period of the COVID 19 pandemic. The study is multicenter retrospective from October 2018 to May 2022, including 380 cases of hospitalised epistaxis, with the mention that March 2020 is considered the beginning of the pandemic period. 60.8% of the patients enrolled in the study in the pre-pandemic period (60.8%) and 39.2% in the pandemic period. Differences between groups were not statistically significant between study entries (pre-pandemic vs. pandemic) and age (p=0.331), gender (p=0.916) or existence of local causes for epistaxis (p=0.895). Patients with general causes for epistaxis were more frequently enrolled in the pandemic period, while patients without general causes for epistaxis were more frequently enrolled in the pre-pandemic period. Patients with a hospitalisation period of more than 5 days were more frequently enrolled in the pre-pandemic period while patients with a hospitalisation period of 3 to 5 days were more frequently enrolled in the pandemic period. Also, patients with idiopathic epistaxis were more frequently enrolled in the pre-pandemic period. Based on the results presented in our study, the period of the Covid 19 pandemic directly influenced both the number of patients and the period of hospitalisation.

Current approach of juvenile nasopharyngeal angiofibroma: a case series.

Juvenile nasopharyngeal angiofibroma (JNA) is a rare benign tumor that affects predominantly males and is known by its highly vascular character. We have performed a 3-year retrospective study of patients with JNA surgically treated within the third ENT Department of Prof. Dr. Dorin Hociota Institute of Phonoaudiology and Functional ENT Surgery, Bucharest, Romania. In all the cases, the patients were investigated both clinically and through medical imaging before surgery and all tumors were embolized. Our study comprised of eight cases, of which seven were solved by endoscopic endonasal approach and one case was treated through a combined endonasal-external approach. JNA should always be managed through a multidisciplinary team (MDT) approach in centers with adequate experience, to gain favorable results.

Radiofrequency EMF irradiation effects on pre-B lymphocytes undergoing somatic recombination.

Intense electromagnetic fields (EMFs) induce DNA double stranded breaks (DSBs) in exposed lymphocytes.We study developing pre-B lymphocytes following V(D)J recombination at their Immunoglobulin light chain loci (IgL). Recombination physiologically induces DNA DSBs, and we tested if low doses of EMF irradiation affect this developmental stage. Recombining pre-B cells, were exposed for 48 h to low intensity EMFs (maximal radiative power density flux S of 9.5 µW/cm2 and electric field intensity 3 V/m) from waves of frequencies ranging from 720 to 1224 MHz. Irradiated pre-B cells show decreased levels of recombination, reduction which is dependent upon the power dose and most remarkably upon the frequency of the applied EMF. Although 50% recombination reduction cannot be obtained even for an S of 9.5 µW/cm2 in cells irradiated at 720 MHz, such an effect is reached in cells exposed to only 0.45 µW/cm2 power with 950 and 1000 MHz waves. A maximal four-fold recombination reduction was measured in cells exposed to 1000 MHz waves with S from 0.2 to 4.5 µW/cm2 displaying normal levels of γH2AX phosphorylated histone. Our findings show that developing B cells exposure to low intensity EMFs can affect the levels of production and diversity of their antibodies repertoire.

The recesses of the retro-tympanum.

OBJECTIVE: To study the anatomy of the posterior tympanic wall and to describe the four recesses (Sinus Tympani-ST, Lateral Tympanic Sinus-LTS, Posterior Tympanic Sinus-PTS, Facial Recess-FR) with their variations in shape, dimension, internal configuration. MATERIAL AND METHODS: Thirty-seven cadaver temporal bones were examined through the anterior dissection approach. The opening was enlarged by drilling to allow a good microscopic or rigid endoscopic view to the retro-tympanum. Measurements of the recesses were made with gradually millimeter wire, dial micrometric indicator (comparator clock), various right angled picks and sometimes by means of cast-impression--used in stomatology for dental impression (interior configuration). RESULTS: Out of 37 specimens, we have found PTS in 64.86% (24 cases), ST in 94.59% (35 cases), and LTS in 59.45% (22 cases). For FR, we have found four types. The most constant cell, located in the inferior external part of FR and in the same time, the biggest one was suprapyramidal Sappey's fossa, which we have found in 75.67% (28 cases) of all specimens. Sinus entrance of each recess was very variable, but in the most cases, we have found that the long diameter of ST and LTS was usually directed vertically and of PTS usually horizontally. The deeper recess was ST-2.74 mm (range 0.5 to 6.2 mm). Pyramidal eminence was visualized in 100% of cases, the pyramidal ridge in 89.18%, the chordal ridge in 78.37%, the suprapyramidal ridge in 35.13%, ponticulus in 83.78% and subiculum in 75.67%. In many cases, the inner configuration of each sinus was irregular, presenting either small recesses (0.2 to 1 mm) under the bone borders at the entrance in the sinuses or communication between the two sinuses. CONCLUSIONS: Presence of the all four recess is not a rule. Marked variation in the size and shape and especially in the internal configuration of all recesses is a rule. All recesses of the retro-tympanum can be recognized more easily, if we identify at first all elements of positive projection (pyramidal eminence, chordal ridge, styloid eminence, styloid ridge, pyramidal ridge, suprapyramidal ridge, ponticulus, subiculum).

Involvement of inflammatory cells in chronic rhinosinusitis with nasal polyps.

Inflammation plays an important role in the pathogenesis of nasal polyps. Understanding the biomolecular action mechanisms of inflammatory elements can contribute to improving the prognosis of these lesions. The study analyzed the distribution and immunohistochemically quantified eosinophils [eosinophil major basic protein (BMK-13)], lymphocytes [cluster of differentiation (CD) 4, CD8, CD20] and plasmocytes (CD138) in both the epithelial and stromal compartment in relation to composite scores, which included specific histopathological parameters for 50 sinonasal polyps. Inflammatory elements predominated at stromal level, the high histological composite scores being frequently associated with increased expression of inflammatory elements. Also, the numerical distribution of inflammatory elements indicated positive linear relations within the groups BMK-13∕CD8 and CD4∕CD20∕CD138, and a negative linear relation between the two groups. This aspect can support the existence of alternative or sequential pathogenic mechanisms involved in the pathogenesis of sinonasal polyps, and the results obtained can be used for a better stratification of patients in order to optimize the therapy.

Comparative clinicopathological aspects of chronic tonsillitis and adenoiditis in children.

Chronic palatal and nasopharyngeal inflammations are common lesions in pediatric pathology, with major effects on children's development. The study included 34 cases of chronic tonsillitis and adenoiditis for which we quantified immunohistochemically and analyzed the distribution of inflammatory elements in the follicular, extrafollicular and epithelial compartments, in relation to the composite histological scores and the clinico-epidemiological profile of the lesions. The cases were more frequent under the age of 10, in female patients, coming from urban areas, with the diagnosis of tonsillitis. B-lymphocytes have been associated with follicular areas in tonsillitis and epithelial areas in adenoiditis. In all compartments, T-lymphocytes were more frequently associated with tonsillitis and plasma cells associated with adenoiditis. Macrophages and dendritic cells had a relatively uniform distribution for the three compartments in all cases. The results obtained indicate different inflammatory phenotypes for chronic tonsillitis and adenoiditis, an aspect that may be useful for stratifying patients for optimal therapy.

Clinical-morphological profiles of esophageal carcinoma's main types.

AIM: The purpose of the study was to define and then to compare particularly the morphological profiles of the main morphological types of esophageal carcinoma (EC). PATIENTS, MATERIALS AND METHODS: The studied group included 46 operated EC patients. Few parameters were clinical (gender and age). The rest of them described both gross and histological features of the entire group and of the two main histological types of carcinoma (lesion' site, lateral extension, lesion dimensions, gross aspect, and histological type, and tumor grade, and stage). Stratification scales of cases were defined according to each parameter in order to compare the data and a statistical apparatus [Student's t-test and χ² (chi-squared) test] was used. RESULTS: The studied tumors were encountered mostly in mature adult and elderly men, usually in the lower segments of the esophagus. Many of them had between five and ten cm in the long diameter and produced stenosis. Most of them had infiltrating appearance combined often with protruding or∕and ulcerated aspects. Usually, the tumors were poorly differentiated and in stage III. The two main histological types of EC showed different morphological profiles. Data from the literature revealed sometimes wide ranges of variation for the studied morphological parameters. Our results were within these ranges of variation. CONCLUSIONS: ECs proved to be aggressive and late diagnosed tumors in general, with distinct morphological and behavioral profiles for the two main histological types. Comparisons with literature data confirmed many of our observations regarding the clinical and morphological aspects of both ECs as a whole and its histological types.

Clinical and histopathological aspects in otomastoiditis.

We aimed to establish correlations between the clinical manifestations and histopathological changes occurring in otomastoiditis. To this effect, we have made a retrospective clinical and statistical study on a number of 146 patients diagnosed with otomastoiditis, admitted in the Otorhinolaryngology Clinic of the Emergency County Hospital, Craiova, during 2003-2007. Of the 146 patients, 128 (87.67%) were suggested to follow a radical surgical treatment (total petromastoidian evidation), in 10 cases (6.85%) a partial petromastoidian evidation was performed, and in eight cases (5.48%) there were made reappraisals of the petromastoidian evidation cavities. When the surgical procedures were performed, there were collected fragments of mucous from the middle ear or from the mastoid cavities, which were sent for examination to the Laboratory of Anatomical Pathology, where there were performed classical histological (Hematoxylin-Eosin and trichromic Van Gieson) and immunohistochemistry staining. The clinical and statistical study emphasized the fact that the onset of the disease was chronic in 143 patients (97.95%) and of acute type in three patients (2.05%). Taking into account the medical history of the patients, repeated episodes and incorrectly treated of acute infections of the middle ear (otitis) were found in most cases, amounting to a percentage of 97.95%. The distribution of the patients on the five years was relatively uniform, in 2003 - 24 cases (16%), in 2004 - 36 cases (25%), in 2005 - 28 cases (19%), in 2006 - 25 cases (17%), in 2007 - 33 cases (23%), and the most affected age groups were 21-30-year-old (19.86%) and 51-60-year-old (21.92%). Analyzing the cases included in the studied group emphasized the fact that for a relatively large percentage of patients (87.67%) it was required as an immediate necessity the surgical procedure of total petromastoidian evidation, due to the extensive damages, the negligence of the patients that did not contact in proper time a medical specialist. The most frequent histopathological diagnose was of cholesteatoma in 60 cases (41.10%), fact which also grounded the surgical procedure. Immunohistochemical and microscopic study highlighted in the epithelium areas of necrosis, associated with areas of reactive hypertrophy and hyperplasia, and in the chorion, the existence of a chronic inflammatory infiltrate made up of lymphocytes, plasmocytes and macrophages. The immunohistochemistry studies stressed the preponderant presence of T-lymphocytes and macrophages in the chorion of the mucous of the middle ear, confirming the presence of an immune reaction, predominantly of cellular type.

Malignant fibrous histiocytoma of larynx with giant cell: case report and histological-clinical considerations.

INTRODUCTION: Malignant fibrous histiocytoma (MFH) is a tumor which has a mesenchymal origin, and an uncertain histogenesis. MFH with giant cell accounts for 3-15% of all malignant fibrous histiocytomas. OBJECTIVE: To explore the histopathology, the diagnosis and the treatment of MFH with giant cell of larynx. PATIENT AND METHODS: We report a case of a glottic MFH at a 59-year-old male, who has been smoking for 30 years, and was hospitalized in June 2008 at Emergency County Hospital of Craiova. RESULTS: The largely-sized tumor was originated in glottic area, upper a left vocal cord, with no paresis. The tumor determined respiratory failure and dysphonia. Patient underwent surgical excision of the tumor after an emergency tracheotomy. The immunohistochemical techniques proved positive for vimentin, smooth muscle actin, CD68, CD34, bcl2, EGFR, S100, Ki67, and negative for CD117, NFT, chromogranin, c-erbB2, CK34betaE12, MNF116, and p53. CONCLUSIONS: Malignant fibrous histiocytomas (MFH) with giant cell of larynx are very rare mesenchymal neoplasm (this case seems the first reported). The diagnosis of MFH of larynx was difficult and the immunohistochemistry could have been helpful.

The Design of New HIV-IN Tethered Bifunctional Inhibitors Using Multiple Microdomain Targeted Docking.

Currently, used antiretroviral HIV therapy drugs exclusively target critical groups in the enzymes essential for the viral life cycle. Increased mutagenesis of their genes changes these viral enzymes, which once mutated can evade therapeutic targeting, effects which confer drug resistance. To circumvent this, our review addresses a strategy to design and derive HIV-Integrase (HIV-IN) inhibitors which simultaneously target two IN functional domains, rendering it inactive even if the enzyme accumulates many mutations. First we review the enzymatic role of IN to insert the copied viral DNA into a chromosome of the host T lymphocyte, highlighting its main functional and structural features to be subjected to inhibitory action. From a functional and structural perspective we present all classes of HIV-IN inhibitors with their most representative candidates. For each chosen compound we also explain its mechanism of IN inhibition. We use the recently resolved cryo EM IN tetramer intasome DNA complex onto which we dock various reference IN inhibitory chemical scaffolds such as to target adjacent functional IN domains. Pairing compounds with complementary activity, which dock in the vicinity of a IN structural microdomain, we design bifunctional new drugs which may not only be more resilient to IN mutations but also may be more potent inhibitors than their original counterparts. In the end of our review we propose synthesis pathways to link such paired compounds with enhanced synergistic IN inhibitory effects.

Chronic tonsillitis: histological and immunohistochemical aspects.

OBJECTIVES: Chronic tonsillitis represents the most common inflammatory lesions of the pharynx determining numerous local or distant evolutive complications. We decided to study the histological and especially immunohistochemical expression of this pathology. MATERIAL AND METHODS: We have studied 112 surgical samples representing tonsils resected from 56 patients with chronic tonsillitis. The tonsillectomies were performed in the ENT Clinic of the Emergency County Hospital of Craiova, between 01.01.2007--31.12.2007. The processed histological samples were stained using Hematoxylin-Eosin, light green trichromic and argental impregnation. For the immunohistochemical study, we used LSAB method with CD20, CD45 RO, CD68 antibodies in order to reveal and differentiate T- and B-lymphocytes and also the macrophages. RESULTS: In all samples, we found hyperplasia and hypertrophy of the lymphoid follicle with excessive developing of the clear germinal center as a normal reaction to antigens presence. In some cases, we remarked micro hemorrhages and hematic extravasations inside the follicles, probably due to the excessive virulence of the pathogens causing endothelial lesions. The conjunctive stroma was enriched in collagen fibbers, in some cases organized in strong fascicles with an obvious tendency to divide the tonsils in lobules. The young fibroblastic type cells were numerous. The specific reticulin fibbers had a low representation being disorganized. The immunohistochemical study proved that the clear center of the lymphoid follicles was occupied by B-lymphocytes, but the T-lymphocytes were present in the cortical region of the follicles, perifollicles and in the surface epithelium. CONCLUSIONS: In some pathological cases, the predominant cellular population of the clear center was formed by T-lymphocytes.

Transglutaminase mediated microencapsulation of sea buckthorn supercritical CO2 extract in whey protein isolate and valorization in highly value added food products.

Sea buckthorn carotenoids extracted using CO2 supercritical fluids method were encapsulated within whey proteins isolate by transglutaminase (TG) mediated crosslinking reaction, coacervation and freeze drying. The encapsulation efficiency was 36.23 ±â€¯1.58%, with ß-carotene the major carotenoid present in the powder. The confocal analysis revealed that TG-ase mediated cross-linking reaction enhanced the complexes stability to such a manner that a double microencapsulation was performed. The powder showed an antioxidant activity of 2.16 ±â€¯0.14 mMol Trolox/g DW and an antifungal activity against Penicillium expansum MIUG M11. Four variants of domestic ice creams were obtained, with a total carotenoids content variation of 1.63 ±â€¯0.03 mg/g D.W. in sample with 2% powder and 6.38 ±â€¯0.04 mg/g D.W. in samples with 4% extract, having satisfactory antioxidant activity. The storage stability test showed a decrease in both total carotenoids content and antioxidant activity in all samples during 21 days at -18 °C. A protective effect of microencapsulation was evidenced.

Clinical, morphological and immunohistochemical aspects in laryngeal premalignant lesions.

OBJECTIVE: We investigated the proliferative activities in premalignant laryngeal vocal chord lesions treated by epithelial stripping in microlaryngoscopy, using immunohistochemical staining with anti-p53, anti-PCNA and anti-Ki-67 monoclonal antibody and we correlated with clinic and morphologic aspects. MATERIAL AND METHODS: The study was made on 32 patients hospitalized in Craiova ENT Clinic in 2005 presenting lesions precursor to vocal cord malignity. The lesion's aspect was observed using suspended microlaryngoscopy, a biopsy was performed and biological tests were examined from a pathological and immunohistochemical point of view, with the investigation of the following immunohistochemical markers: Ki-67, PCNA and p53. RESULTS: 13 cases (41%) presented red hypertrofic cronic laryngitis, seven cases (22%) presented white hypertrofic cronic laryngitis, and 12 cases (37%) presented papillomas with with simple, moderate, severe dysplasia and in situ carcinoma in 62.5%, 22%, 12.5% and, respectively, 3% of the cases. All the dysplasic lesions, no matter the dysplasic degree, have presented alteration of both surface eplitelium and chorion. The expression of Ki-67, PCNA and p53 was correlated with the dysplasia's degree in various proportions. CONCLUSIONS: In clinical practice, morphological grading of dysplasia is difficult to evaluate. The Ki-67 and PCNA markers were correlated with the dysplasia degree; the expression of p53 was present only in 28% cases with moderate dysplasia and in one case with in situ carcinoma.

TNF-α evaluation in tonsil cancer.

Squamous cell tonsil carcinoma is the most frequent form of oropharyngeal cancer, representing 70-80% of the total of head and neck malignant tumors. Poor clinical symptoms make that 60-80% of patients with squamous cell tonsil carcinoma have a late diagnosis, in the third and fourth stages, when the tumor exceeds the organ limits, invading the pharyngeal wall or the tongue base, being associated with metastases in the laterocervical lymphatic ganglions. The tumor necrosis factor alpha (TNF-α) represents an important inflammation mediator associated to carcinogenesis and even to tumor progression. We evaluated the seric values of TNF-α in a group of patients with tonsil cancer in comparison to a group of patients with chronic tonsillitis, as well as the reaction of mastocytes and macrophages in the two types of tonsil lesions. Seric levels of TNF-α in squamous cell tonsil carcinoma were quite high, varying from 1000 to 2000 pg÷mL, and in four patients, with poorly differentiated tonsil carcinoma in the fourth stage, the TNF-α values varied from 2000 to 4000 pg÷mL. In the patients undergoing radiotherapy, the TNF-α seric levels were within normal limits. In chronic tonsillitis, the TNF-α seric level varied from 10 to 200 pg÷mL. There were not observed any significant differences between the two types of tonsil lesions, regarding the macrophages and mast cells density on the surface unit.

Chondrosarcoma of the hyoid bone: a case report.

Chondrosarcoma is a malignancy of the mesenchymal tissue derived from transformed cells that produce the cartilage matrix. In the neck area, it represents less than 0.5% of malignant tumor pathology. Chondrosarcoma of the hyoid bone is extremely rare, only 20 cases having been published so far (PubMed 2014). We present the case of a 30-year-old patient from the urban area, admitted in the ENT (Ear, Nose & Throat) Emergency Service with inspiratory dyspnea, dysphagia, stomatolalia, with evolutive and progressive clinical history of 2-3 months. Endoscopic examination revealed a pharyngolaryngeal tumor process located in the right vallecula, who by mass effect displaces the above-hyoid epiglottis. CT (computerized tomography) scan described a cervical polycystic tumor aspect, with multiple septae and inside calcifications with a diameter of 3-4 mm. Surgery consisted in removal of the tumor process together with the hyoid bone. Histopathological and especially immunohistochemical examination established the diagnosis of low-grade chondrosarcoma of the hyoid bone. For assessment of the phenotype of the tumor cells, the following immunohistochemical markers were used: p53, Ki67. The patient followed radiochemotherapic oncological treatment and returned for regular follow-ups. There was a positive development with no signs of regional or remote relapse or metastasis for 24 months after surgical treatment. Surgery is the treatment of choice, with complete removal of the tumor, with chemoradiation playing an adjuvant role. Regular tracking of the patient is mandatory.

Advances in structure-function relationships of tyrosinase from Agaricus bisporus - investigation on heat-induced conformational changes.

A combination of fluorescence spectroscopic measurements, inactivation kinetics and in silico prediction was used in the present study to investigate the heat induced behaviour of tyrosinase from Agaricus bisporus. The phase diagram indicated the existence of at least two distinct species induced by the temperature increase up to 75°C. Regardless of calcium ion presence, the fluorescence intensity results suggest that tyrosinase tends to form aggregates after 10min at 75°C. The quenching experiments using acrylamide and iodide demonstrate a more flexible conformation of tyrosinase at higher temperature. Detailed insights into tyrosinase structure after performing molecular dynamics simulations, suggest important structural rearrangements of the protein with the temperature increase. The copper coordinating His(94) residue was predicted to be involved in salt bridge formation with Glu(98), therefore causing significant alteration of the substrate binding site with increasing temperature. These significant changes in tyrosinase structure at temperatures over 60°C might lead to enzyme inactivation.

DNA bending in the synaptic complex in V(D)J recombination: turning an ancestral transpososome upside down.

In all jawed vertebrates RAG (recombination activating gene) recombinase orchestrates V(D)J recombination in B and T lymphocyte precursors, assembling the V, D and J germline gene segments into continuous functional entities which encode the variable regions of their immune receptors. V(D)J recombination is the process by which most of the diversity of our specific immune receptors is acquired and is thought to have originated by domestication of a transposon in the genome of a vertebrate.  RAG acts similarly to the cut and paste transposases, by first binding two recombination signal DNA sequences (RSSs), which flank the two coding genes to be adjoined, in a process called synaptic or paired complex (PC) formation. At these RSS-coding borders, RAG first nicks one DNA strand, then creates hairpins, thus cleaving the duplex DNA at both RSSs. Although RAG reaction mechanism resembles that of insect mobile element transposases and RAG itself can inefficiently perform intramolecular and intermolecular integration into the target DNA, inside the nuclei of the developing lymphocytes transposition is extremely rare and is kept under proper surveillance. Our review may help understand how RAG synaptic complex organization prevents deleterious transposition. The phosphoryl transfer reaction mechanism of RNAseH-like fold DDE motif enzymes, including RAG, is discussed accentuating the peculiarities described for various transposases from the light of their available high resolution structures (Tn5, Mu, Mos1 and Hermes). Contrasting the structural 3D organization of DNA in these transpososomes with that of the RSSs-DNA in RAG PC allows us to propose several clues for how evolutionarily RAG may have become "specialized" in recombination versus transposition.