Selectivity of Ni(II) and Zn(II) binding to Sporosarcina pasteurii UreE, a metallochaperone in the urease assembly: a calorimetric and crystallographic study

Urease is a nickel-dependent enzyme that plays a critical role in the biogeochemical nitrogen cycle by catalyzing the hydrolysis of urea to ammonia and carbamate. This enzyme, initially synthesized in the apo form, needs to be activated by incorporation of two nickel ions into the active site, a process driven by the dimeric metallochaperone UreE. Previous studies reported that this protein can bind different metal ions in vitro, beside the cognate Ni(II). This study explores the metal selectivity and affinity of UreE from Sporosarcina pasteurii (Sp, formerly known as Bacillus pasteurii) for cognate [Ni(II)] and noncognate [Zn(II)] metal ions. In particular, the thermodynamic parameters of SpUreE Ni(II) and Zn(II) binding have been determined using isothermal titration calorimetry. These experiments show that two Ni(II) ions bind to the protein dimer with positive cooperativity. The high-affinity site involves the conserved solvent-exposed His¹⁰⁰ and the C-terminal His¹⁴⁵, whereas the low-affinity site comprises also the C-terminal His¹⁴⁷. Zn(II) binding to the protein, occurring in the same protein regions and with similar affinity as compared to Ni(II), causes metal-driven dimerization of the protein dimer. The crystal structure of the protein obtained in the presence of equimolar amounts of both metal ions indicates that the high-affinity metal binding site binds Ni(II) preferentially over Zn(II). The ability of the protein to select Ni(II) over Zn(II) was confirmed by competition experiments in solution as well as by analysis of X-ray anomalous dispersion data. Overall, the thermodynamics and structural parameters that modulate the metal ion specificity of the different binding sites on the protein surface of SpUreE have been established.     

The Impact of Short-Term,Intensive Antifolate Treatment (with Pyrimethamine and Sulfadoxine) and Antibiotics Followed by Long-Term,Secondary Antifolate Prophylaxis on the Rate of Toxoplasmic Retinochoroiditis Recurrence

PurposeTo assess the impact of intensive antifolate treatment, followed by secondary antifolate prophylaxis (A-SP) on the recurrence rate of toxoplasmic retinochoroiditis (TRC). To investigate whether there are any other factors potentially predisposing for recurrence.ResultsWhen secondary antifolate prophylaxis (A-SP) was instituted immediately after the treatment for TRC, the probability of 3-year recurrence–free survival after the first course of A-SP was 90.9%. A recurrence was most likely approximately 3.5 years after the first treatment. A univariate Cox regression model demonstrated that a risk for recurrence was 2.82 times higher (p = 0.02) in patients with retinal scars. In the multivariate analysis, the risk for recurrence was 2.41 higher (p = 0.06). In patients with haemorrhagic lesions the risk for recurrence was lower, aRR = 0.17 (approaching borderline statistical significance p = 0.08).ConclusionsWith the institution of A-SP of immediately after the intensive treatment for TRC, i.e. when a reactivation was most likely, there was no recurrence during A-SP. Following A-SP the recurrence rates were low and recurrence-free periods tended to be longer. The treatment regimen employed had a beneficial effect on the recurrence interval as it reduced and delayed the highest probability of recurrence.     

Investigating DNA Radiation Damage Using X-Ray Absorption Spectroscopy

The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA.     

The importance of stem cell engineering in head and neck oncology

Head and neck squamous cell carcinoma is the sixth leading cause of cancer worldwide. The most common risk factors are carcinogens (tobacco, alcohol), and infection of the human papilloma virus. Surgery is still considered as the treatment of choice in case of head and neck cancer, followed by a reconstructive surgery to enhance the quality of life in the patients. However, the widespread use of artificial implants does not provide appropriate physiological activities and often cannot act as a long-term solution for the patients. Here we review the applicability of multiple stem cell types for tissue engineering of cartilage, trachea, vocal folds and nerves for head and neck injuries. The ability of the cells to self-renew and maintain their pluripotency state makes them an attractive tool in tissue engineering.     

Anticonvulsant Activity of Enantiomeric N‐trans‐Cinnamoyl Derivatives of 2‐Aminopropan‐1‐ols and 2‐Aminobutan‐1‐ols

Epilepsy, one of the most frequent neurological disorders, is still insufficiently treated in about 30% of patients. As a consequence, identification of novel anticonvulsant agents is an important issue in medicinal chemistry. In the present article we report synthesis, physicochemical, and pharmacological evaluation of N‐trans‐cinnamoyl derivatives of R and S‐2‐aminopropan‐1‐ol, as well as R and S‐2‐aminobutan‐1‐ol. The structures were confirmed by spectroscopy and for derivatives of 2‐aminopropan‐1‐ols the configuration was evaluated by means of crystallography. The investigated compounds were tested in rodent models of seizures: maximal electroshock (MES) and subcutaneous pentetrazol test (scPTZ), and also in a rodent model of epileptogenesis: pilocarpine‐induced status prevention. Additionally, derivatives of 2‐aminopropan‐1‐ols were tested in benzodiazepine‐resistant electrographic status epilepticus rat model as well as in vitro for inhibition of isoenzymes of cytochrome P450. All of the tested compounds showed promising anticonvulsant activity in MES. For R(–)‐(2E)‐N‐(1‐hydroxypropan‐2‐yl)‐3‐phenylprop‐2‐enamide pharmacological parameters were found as follows: ED₅₀ = 76.7 (68.2–81.3) mg/kg (MES, mice i.p., time = 0.5 h), ED₅₀ = 127.2 (102.1–157.9) mg/kg (scPTZ, mice i.p., time = 0.25 h), TD₅₀ = 208.3 (151.4–230.6) mg/kg (rotarod, mice i.p., time = 0.25 h). Evaluation in pilocarpine status prevention proved that all of the reported compounds reduced spontaneous seizure activity and act as antiepileptogenic agents. Both enantiomers of 2‐aminopropan‐1‐ols did not influence cytochrome P450 isoenzymes activity in vitro and are likely not to interact with CYP substrates in vivo. Chirality 28:482–488, 2016. © 2016 Wiley Periodicals, Inc.     

Fibrous Aggregates of Short Peptides Containing Two Distinct Aromatic Amino Acid Residues

The aim of the study was the assessment of the ability of short peptides to form aggregates under physiological conditions. The dipeptides studied were derived from different aromatic amino acids (heteroaromatic peptides). Tripeptides were obtained from two distinct aromatic amino acids and cysteine or methionine residue in the C‐terminal, N‐terminal, or central position. The ability of the peptides to form fibrous aggregates under physiological conditions was evaluated using three independent methods: the Congo Red assay, the Thioflavin T assay, and microscopic examinations using normal and polarized light. Materials potentially useful for regenerative medicine were selected based on their cytotoxicity to the endothelial cell line EA.hy 926 and physicochemical properties of films formed by peptides. The required parameters of biocompatibility were fulfilled by H?PheCysTrp?OH, H?PheCysTyr?OH, H?PheTyrMet?OH, and H?TrpTyr?OH.     

Assessment of cartilage invasion in case of laryngeal cancer by means of longitudinal sectioning for histopathology – Clinical implications

AimThe aim of the study was to assess the accuracy of radiological diagnosis of laryngeal cartilage infiltration by histopathological examination of laryngeal specimen after total laryngectomy.BackgroundDespite the development of new medical technologies and significant clinical advances allowing early diagnosis and treatment of laryngeal cancer, mortality is still on the rise. Neoplastic infiltration of the laryngeal cartilages is the most common source of error in the assessment of cancer staging. Furthermore, cartilage invasion is listed as a contraindication to partial surgical techniques as well as radiotherapy.Materials and methodsThe study was carried out on 21 larynges following total laryngectomy. Before taking the decision to perform surgery, high-resolution CT scans were performed in all cases. An extended histopathological examination was conducted using a unique vertical cross-section of the whole larynx.ResultsPathology reported 2 cases of arytenoid cartilage invasion, 5 cases of cricoid cartilage invasion, 12 cases of thyroid cartilage penetration, 1 case of internal cortex invasion and 9 cases of extra-laryngeal spread. CT imaging identified 8 of 13 cases (61.5%) of pathologically proven invasion of thyroid cartilage and only 2 cases (2/9, 22%) of extra-laryngeal spread. According to CT results, arytenoid cartilage invasion was correctly identified in 2 cases, cricoid cartilage invasion in 4 (4/5, 80%). The positive predictive values for thyroid, cricoid and arytenoid cartilage invasion and penetration were 80%, 66.7% and 50%, respectively. In case of pre-laryngeal spread the positive predictive value was 100%.ConclusionDespite increasingly advanced methods involved in the diagnosis of laryngeal cancer, many discrepancies may be observed between the radiological and histopathological assessments. CT imaging has limitations especially in thyroid cartilage penetration and extra-laryngeal spread assessment in advanced laryngeal cancer cases. An extended histopathological examination, involving vertical cross-sections of the whole larynx is a very precise study that allows a precise determination of local cancer staging (T).