Direct evidence for the alterations in protein structure and conformation upon in vitro nonenzymatic glycosylation.

The formation of nonenzymatic glycosylation products appears to be a link between chronic hyperglycaemia and long-term diabetic complications. However, little is known concerning the glycation-induced modifications in the structure and conformation of proteins, which possibly underlie their altered functional characteristics. This study conveys a direct evidence for and compares the glucose-induced modifications in the conformation of three proteins with various half-lives: bovine serum albumin, human haemoglobin and bovine tendon collagen. These proteins incubated in vitro with glucose in various media containing optionally EDTA and Fe2+ ions contained up to 4-10 times as much attached glucose as did their relevant controls, and the extent of glycation was the highest in the samples incubated under air or in the absence of EDTA. The fluorescence and ESR data indicate that the Trp in albumin molecule, given albumin glycation-induced structural modifications, became more exposed to water surrounding solution whereas the Trp residues of haemoglobin remained shielded from water; also collagen fluorescence derived from the supposedly newly formed covalent crosslinks is vastly increased, and particularly when collagen was glycated under air or in the presence of Fe2+ ions. Possible mechanisms underlying the increased mobility of selected protein domains and glycation-mediated alterations in protein conformation are considered and discussed.     

Detection of plasmid-mediated quinolone resistance in clinical isolates of Enterobacteriaceae strains in Hamadan,West of Iran

Plasmid mediated quinolone resistance (PMQR) determinants have arisen as a significant concern in recent years. The aim of this study was screening of resistant-clinical isolates to fluoroquinolone antibiotics and detection of qnr and aac(6′)-Ib-cr genes.For this purpose we collected 100 fluoroquinolone-resistant Enterobacteriaceae which were from 3 hospitals in Hamadan, west provinces of Iran, between October 2012 and June 2013. The all samples were identified by biochemical tests and confirmed by PCR method. Antimicrobial susceptibility to 14 antimicrobial agents including levofloxacin and ciprofloxacin were determined by disk diffusion methods and ciprofloxacin MIC was obtained by broth microdilution method as Clinical Laboratory Standards Institute (CLSI) recommendations. The isolates were screened for the presence of qnrA, qnrB, qnrS and aac(6′)-Ib-cr genes using PCR assay. Among the screened isolates, 64 strains (64%) of Escherichia coli, 23 strains (23%) of Klebsiella pneumoniae, 13 strains (13%) of Proteus mirabilis were collected as quinolone-resistant isolates. out of 100 isolates, two (2%) were positive for qnrS, seventeen (17%) isolates were positive for qnrB and we did not find qnrA gene in any of the isolates. There were also 32 positive isolates for aac(6′)-Ib-cr determinant. We described the prevalence of qnr and aac(6′)-Ib-cr genes in fluoroquinolone-resistant Enterobacteriaceae in Hamadan city. The carriage rate of multidrug-resistant Enterobacteriaceae in healthy people in Hamadan City is extremely high. Moreover, genes encoding transferable quinolones, in particular aac(6′)-Ib-cr, are highly prevalent in these strains.     

Frequency of genetic polymorphisms of ADAM33 and their association with allergic rhinitis among Jordanians

Allergic rhinitis is a chronic inflammatory disease that is assumed to be due to an interaction between different genetic and/or environmental factors. A disintegrin and metalloprotease domain 33 (ADAM33) has been extensively studied as a susceptibility gene in asthma and has been linked to bronchial hyper-responsiveness. In this study, we investigated the association between ADAM33 single nucleotide polymorphisms and the incidence of allergic rhinitis among the Jordanian population. We conducted a case–control association study on 120 adult individuals diagnosed with allergic rhinitis and 128 normal healthy controls. 8 single-nucleotide polymorphisms in ADAM33 were genotyped using PCR-RFLP method. No significant differences in the allelic frequencies of all SNPs tested between AR patients and the control volunteers were found, although S2 C/G SNP showed a tendency toward significance with P = 0.06. On the genotype level significant association were found in the following genotypes: T1 AA, T1 AG, T2 GG, T2 AG, T + 1 GG, T + 1 AG, V4 CG, S2 CC, S2 CG, Q-1AA. Seven haplotypes were present only within AR patients and eight haplotypes were completely absent from the AR patients. Three haplotypes exhibited significant association with AR P ≤ 0.05, two of them were present only in AR patients. In conclusion, the polymorphisms in the ADAM33 gene are associated with susceptibility to AR in the Jordanian population. Furthermore, the haplotype of the tested SNPs were also associated with the risk of AR.     

Cooperative Use of VCD and XRD for the Determination of Tetrahydrobenzoisoquinolines Absolute Configuration: A Reliable Proof of Memory of Chirality and Retention of Configuration in Enediyne Rearrangements

The absolute configurations (AC) of azaheterocylic compounds resulting from the cascade rearrangement of enediynes involving only light atoms were unambiguously assigned by the joint use of vibrational circular dichroism (VCD) and copper radiation single crystal X‐ray diffraction (XRD). These AC determinations proved that the rearrangements of enediynes proceeded with memory of chirality and retention of configuration. Chirality 25:832–839, 2013. © 2013 Wiley Periodicals, Inc.     

In vitro behavior and UV response of melanocytes derived from carriers of CDKN2A mutations and MC1R variants

Coinheritance of germline mutation in cyclin‐dependent kinase inhibitor 2A (CDKN2A) and loss‐of‐function (LOF) melanocortin 1 receptor (MC1R) variants is clinically associated with exaggerated risk for melanoma. To understand the combined impact of these mutations, we established and tested primary human melanocyte cultures from different CDKN2A mutation carriers, expressing either wild‐type MC1R or MC1RLOF variant(s). These cultures expressed the CDKN2A product p16 (INK4A) and functional MC1R. Except for 32ins24 mutant melanocytes, the remaining cultures showed no detectable aberrations in proliferation or capacity for replicative senescence. Additionally, the latter cultures responded normally to ultraviolet radiation (UV) by cell cycle arrest, JNK, p38, and p53 activation, hydrogen peroxide generation, and repair of DNA photoproducts. We propose that malignant transformation of melanocytes expressing CDKN2A mutation and MC1RLOF allele(s) requires acquisition of somatic mutations facilitated by MC1R genotype or aberrant microenvironment due to CDKN2A mutation in keratinocytes and fibroblasts.     

Fourier transform infrared spectroscopic signature of blood plasma in the progression of breast cancer with simultaneous metastasis to lungs

Despite advanced diagnostic techniques used for detecting cancer, this disease still remains a leading cause of death in the developed world. What is more, the greatest danger for patients is not related with growing of tumor but rather with metastasis of cancer cells to the distant organs. In this study, Fourier transform infrared (FTIR) spectroscopy was used to track chemical changes in blood plasma to find spectral markers of metastatic breast cancer during the disease progression. Plasma samples were taken 1‐5 weeks after orthotropic inoculation of 4T1 metastatic breast cancer cells to mice. The earliest changes detected by FTIR spectroscopy in plasma were correlated with unsaturation of phospholipids and secondary structures of proteins that appeared 2 and 3 weeks, respectively, after 4T1 cells inoculation (micrometastatic phase). Significant alternations in the content and structure of lipids and carbohydrates were identified in plasma at the later stages (macrometastatic phase). When large primary tumors in breast and macrometastases in lung were developed, all bands in FTIR spectra significantly differed from those at earlier phases of the cancer progression. In conclusion, we showed that each phase of the breast cancer progression and its pulmonary metastasis can be characterized by a specific panel of spectral markers.