Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling

A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.     

OPTIMIZATION OF SOLID-STATE FERMENTATION FOR PHYTASE PRODUCTION BY Thermomyces lanuginosus USING RESPONSE SURFACE METHODOLOGY

A strain of Thermomyces lanuginosus, isolated from hot spring water in Turkey, was studied for optimization of phytase production using solid-state fermentation. Effects on fermentation of different production parameters such as substrate type, moisture, culture time, and inoculum size were investigated using a one-factor-at-a-time approach. Central composite design (CCD) of response surface methodology was applied for the optimization of four factors (culture temperature, initial pH, aeration area, age of seeding culture) that were affecting phytase production by Thermomyces lanuginosus in rice bran. Maximum phytase activity was achieved by using rice bran. The optimum levels of variables that supported maximum enzyme activity were moisture 70%, culture time 7 days, inoculum size 40%, culture temperature 55°C, initial pH 7.5, aeration area 30%, age of seeding culture 5 days, sucrose 1%, and ZnSO₄ 2.5 mM. An overall 10.83-fold enhancement in phytase activity (0.30 to 3.248 U) was attained due to the optimization.     

Association Between the Interferon Gamma 874 T/A Polymorphism and the Severity of Valvular Damage in Patients with Rheumatic Heart Disease

Interferon gamma (IFN-γ) is a multifunctional cytokine that plays an important role in modulating almost all phases of the immune response and may be responsible for the increased valvular fibrosis and calcification in the pathogenesis of rheumatic heart disease (RHD). The aim of this study was to investigate the possible relationship between the IFN-γ +874 T/A polymorphism and the severity of valvular damage in the Turkish population. The IFN-γ genotypes were determined in 152 RHD patients and 151 healthy controls by ARMS-PCR. Differences in genotype distribution between patients with RHD and control were evaluated by the χ² test. All statistical analyses were performed with SPSS 15.0 Software program. Frequency of the AA genotype was found to be significantly lower and the TT genotype significantly higher in the RHD group compared to controls (p = 0.002 and p = 0.018, respectively). The TT genotype was found to be significantly higher (26.8% vs. 9.1%, p = 0.009) and the AA genotype significantly lower (29.1% vs. 8.2%, p = 0.001) in the severe valvular disease (SVD) group compared to mild valvular disease group. In the SVD group, 79 patients had mitral balloon valvotomy and/or mitral valve replacement and had significantly higher TT genotype compared to patients with medical follow-up (30.4% vs. 19%, p = 0.001). The data demonstrated that TT genotype is associated with both RHD and the severity of RHD.     

WRN mutations in Werner syndrome patients: genomic rearrangements, unusual intronic mutations and ethnic-specific alterations

Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by null mutations at the WRN locus, which codes for a member of the RecQ family of DNA helicases. Since 1988, the International Registry of Werner syndrome had enrolled 130 molecularly confirmed WS cases from among 110 worldwide pedigrees. We now report 18 new mutations, including two genomic rearrangements, a deep intronic mutation resulting in a novel exon, a splice consensus mutation leading to utilization of the nearby splice site, and two rare missense mutations. We also review evidence for founder mutations among various ethnic/geographic groups. Founder WRN mutations had been previously reported in Japan and Northern Sardinia. Our Registry now suggests characteristic mutations originated in Morocco, Turkey, The Netherlands and elsewhere.     

Predicting bone remodeling around tissue- and bone-level dental implants used in reduced bone width

The objective of this study was to predict time-dependent bone remodeling around tissue- and bone-level dental implants used in patients with reduced bone width. The remodeling of bone around titanium tissue-level, and titanium and titanium–zirconium alloy bone-level implants was studied under 100 N oblique load for one month by implementing the Stanford theory into three-dimensional finite element models. Maximum principal stress, minimum principal stress, and strain energy density in peri-implant bone and displacement in x- and y- axes of the implant were evaluated. Maximum and minimum principal stresses around tissue-level implant were higher than bone-level implants and both bone-level implants experienced comparable stresses. Total strain energy density in bone around titanium implants slightly decreased during the first two weeks of loading followed by a recovery, and the titanium–zirconium implant showed minor changes in the axial plane. Total strain energy density changes in the loading and contralateral sides were higher in tissue-level implant than other implants in the cortical bone at the horizontal plane. The displacement values of the implants were almost constant over time. Tissue-level implants were associated with higher stresses than bone-level implants. The time-dependent biomechanical outcome of titanium–zirconium alloy bone-level implant was comparable to the titanium implant.     

The efficacy of a topical gel with triester glycerol oxide in denture-related mucosal injuries

doi: 10.1111/j.1741‐2358.2011.00549.x
The efficacy of a topical gel with triester glycerol oxide in denture‐related mucosal injuries Objective: The efficacy of a topical gel with triester glycerol oxide for treating ulcerations related to new complete dentures was studied and compared with a placebo gel. Materials and methods: Fifty edentulous patients were enrolled in this double‐blind clinical study. All subjects received new maxillary and mandibular complete dentures and were recalled 1, 3 and 7 days after denture placement. Twenty‐five subjects received topical gel with triester glycerol oxide (TGO), and 25 subjects received topical gel without TGO. At each visit, pain and discomfort with the new dentures was assessed, and at the last visit, healing of ulcerations was also graded by a clinician. Subjects also graded the gel’s taste, smell and ease of use. Results: Specifically, there was no statistically significant difference in ulcer healing (p > 0.05), ratings of smell, taste and ease of use (p > 0.05) or pain and discomfort scores (p > 0.05). However, within‐group differences were observed in pain and discomfort scores between follow‐up sessions (p = 0.01 for all comparisons). Conclusion: Clinically, TGO gel is not effective in the treatment of mucosal injuries occurring following placement of complete dentures.     

Investigating the kinetic mechanism of inhibition of elongation factor 2 kinase by NH125: evidence of a common in vitro artifact

Evidence that elongation factor 2 kinase (eEF-2K) has potential as a target for anticancer therapy and possibly for the treatment of depression is emerging. Here the steady-state kinetic mechanism of eEF-2K is presented using a peptide substrate and is shown to conform to an ordered sequential mechanism with ATP binding first. Substrate inhibition by the peptide was observed and revealed to be competitive with ATP, explaining the observed ordered mechanism. Several small molecules are reported to inhibit eEF-2K activity with the most notable being the histidine kinase inhibitor NH125, which has been used in a number of studies to characterize eEF-2K activity in cells. While NH125 was previously reported to inhibit eEF-2K in vitro with an IC(50) of 60 nM, its mechanism of action was not established. Using the same kinetic assay, the ability of an authentic sample of NH125 to inhibit eEF-2K was assessed over a range of substrate and inhibitor concentrations. A typical dose-response curve for the inhibition of eEF-2K by NH125 is best fit to an IC(50) of 18 ± 0.25 μM and a Hill coefficient of 3.7 ± 0.14, suggesting that NH125 is a weak inhibitor of eEF-2K under the experimental conditions of a standard in vitro kinase assay. To test the possibility that NH125 is a potent inhibitor of eEF2 phosphorylation, we assessed its ability to inhibit the phosphorylation of eEF2. Under standard kinase assay conditions, NH125 exhibits a similar weak ability to inhibit the phosphorylation of eEF2 by eEF-2K. Notably, the activity of NH125 is severely abrogated by the addition of 0.1% Triton to the kinase assay through a process that can be reversed upon dilution. These studies suggest that NH125 is a nonspecific colloidal aggregator in vitro, a notion further supported by the observation that NH125 inhibits other protein kinases, such as ERK2 and TRPM7 in a manner similar to that of eEF-2K. As NH125 is reported to inhibit eEF-2K in a cellular environment, its ability to inhibit eEF2 phosphorylation was assessed in MDA-MB-231 breast cancer, A549 lung cancer, and HEK-293T cell lines using a Western blot approach. No sign of a decrease in the level of eEF2 phosphorylation was observed up to 12 h following addition of NH125 to the media. Furthermore, contrary to the previously reported literatures, NH125 induced the phosphorylation of eEF-2.