Structural studies by high-field NMR spectroscopy of a binary-addressed complementary oligonucleotide system juxtaposing pyrene and perfluoro-azide units

Recently, a new approach has been proposed to improve the site-specificity and efficiency of the modification of nucleic acid target sequences, the binary system of complementary-addressing nucleic acid sequences. The binary system comprises two oligonucleotides, one modified with a photosensitizing group and the other with a photoreactive group. The sites of chemical modification are arranged to bring the two chemical functions close enough together in space to allow efficient energy transfer from the photo-excited photosensitizer to an arylazide moiety which expels N2 to form a nitrene which subsequently covalently labels the target nucleic acid. Structural analysis performed by high-resolution 2D NMR spectroscopy (400 MHz and 600 MHz) are reported for the model binary system 1:2:3, where 1 is the target 12-mer pdGTATCAGTTTCT, 2 is a photoactivatable fluoroazide derivative dAGAAACp-L-Az and 3 is the photosensitizer derivative Pyr-pdTGATAC (here: Az is the p-azidotetrafluorobenzyl group, Pyr the pyrenyl-1-methylamino group, L a linker group). The assignment of oligonucleotide and modifying group protons was performed using 1H COSY, TOCSY and NOESY experiments. Comprehensive analysis of 1H NOESY spectra of 1:2:3 showed that terminal fragments of the complex [5'p-1T-2G-3A-4T-], [-21A-22T-23A-24C], [-8T-9T-10T-11C-12T] and [13A-14G-15A-15A-17A-18C-] gave a continuous set of intra- and inter-nucleotide interactions, typical of regular double-stranded B-DNA. In contrast, the central region of the complex composed of 5C, 6A, 7G, 19T and 20G nucleotide residues, nearest the Pyr and Az groups, was found to be distorted. Thus some signals from aromatic and/or sugar-ring protons of the above nucleotide residues were extremely broadened or almost absent. Moreover, some intra- and/or inter-nucleotide interactions, typical of the regular DNA duplex, were not detected for the [-5C-6A-7G-] and [-19T-20G-] regions of the tandem system. Instead of that, some cross-peaks of low-intensity between the H2 proton of the Pyr group and 7G(H1'), 7G(H2'/H2"), 7G(H3'), 4T(H2"), 4T(H4') and 4T(H5'/H5") were observed. Additional 1H -1H NOE-interactions between methylene protons of the linker group L and some sugar ring protons of 18C nucleotide residue were detected. A preliminary structural model, constructed using proton-proton distances between Pyr and the DNA and Az-L and DNA obtained from a 1H NOESY experiment at 300 ms mixing time as constraints for the refinement of the structure, displayed significant distortion from B-DNA of the double-stranded helix in the middle of the complex, (-5C-6A-7G, -18C-19T-20G-). The Pyr group was located in what remains of the minor groove near 4T, 5C, 6A and 7G and the centroid of the azide ring less than 9A degrees from the centroid of the ring system of Pyr group.     

Hibernoma in the parotid region

Hibernoma is a benign tumour, arising from vestigial remnants of brown adipose cells, which have persisted from embryonic life. Although it is a well recognized entity, it is an extremely rare tumour. Recent reports estimate 81 cases in the world literature, only seven being in the neck. To our knowledge, it has never been reported in the parotid region. We report such a case and discuss the historical background of a hibernoma, along with its pathological and clinical behaviour.     

The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation

The S. cerevisiae SIS1 gene is essential and encodes a heat shock protein with similarity to the bacterial DnaJ protein. At the nonpermissive temperature, temperature-sensitive sis1 strains rapidly accumulate 80S ribosomes and have decreased amounts of polysomes. Certain alterations in 60S ribosomal subunits can suppress the temperature-sensitive phenotype of sis1 strains and prevent the accumulation of 80S ribosomes and the loss of polysomes normally seen under conditions of reduced SIS1 function. Analysis of sucrose gradients for SIS1 protein shows that a large fraction of SIS1 is associated with 40S ribosomal subunits and the smaller polysomes. These and other results indicate that SIS1 is required for the normal initiation of translation. Because DnaJ has been shown to mediate the dissociation of several protein complexes, the requirement of SIS1 in the initiation of translation might be for mediating the dissociation of a specific protein complex of the translation machinery.     

Combination cytotoxic effects of cis-diamminedichloroplatinum(II) and 5-fluorouracil with and without leucovorin against human non-small cell lung cancer cell lines

Both cisplatin (CDDP) and leucovorin (LV) have been shown to enhance cytotoxicity of 5-fluorouracil (FUra) against murine and human neoplasms by increasing intracellular reduced folate concentrations. We were interested in their use in a combination to inhibit non-small cell lung cancer (NSCLC) cell growth and therefore conducted an in vitro study to investigate the cytotoxic activities of combinations of CDDP plus FUra, with and without LV (20 microM), against seven NSCLC cell lines. A tetrazolium assay with application of the classical isobole method was used to test drug combinations. We found that LV enhanced FUra but not CDDP cytotoxicity and that the degree of enhancement was negatively correlated with the effect of FUra. There was an overall additive combination effect of CDDP plus FUra, although there may be synergy at higher effect levels. There was synergy to a combination of CDDP, FUra, and LV, presumably primarily related to the synergistic effects of adding LV to FUra. In summary, LV and CDDP enhanced FUra cytotoxicity in a complementary fashion and there was clear synergy of a combination of CDDP, FUra, and LV against a panel of NSCLC cell lines. Our in vitro results provide a rationale for controlled clinical studies of this three-drug regimen in patients with NSCLC.     

A randomized comparison of the efficacy and toxicity of epirubicin and doxorubicin in the treatment of patients with non-Hodgkin's lymphoma

BACKGROUND: Combination chemotherapy consisting of methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisolone, and bleomycin (MACOP-B) has been frequently used for the treatment of non-Hodgkin's lymphoma. This randomized study was undertaken to assess the efficacy and toxicity of this regimen when either doxorubicin or epirubicin was used as the anthracycline drug. METHODS: Between April 1989 and December 1993, 211 previously untreated patients with intermediate grade and high grade non-Hodgkin's lymphoma were randomized to receive either doxorubicin (n=106) or epirubicin (n=105) with the MACOP-B regimen. These patients were followed through December 1996. Numerous clinical features predictive of response and survival were analyzed. Cardiac and noncardiac toxicity in the two treatment arms were compared. RESULTS: The median age of the patients was 48 years. Complete remission was experienced by 122 patients (58.3%); 62 patients (58.5%) achieved complete remission in the doxorubicin arm and 60 (58.1%) in the epirubicin arm. Response rates, time to treatment failure, relapse data, and overall survival were comparable between the two arms. Morbidity due to mucositis, vomiting, peripheral neuropathy, and cardiotoxicity were also comparable. The overall mortality was 10%. Mortality due to neutropenic sepsis was considerably higher among patients who received epirubicin (10 patients) than among those who received doxorubicin (5 patients). Cardiac evaluation revealed no difference in toxicity between the two arms. CONCLUSIONS: Epirubicin was as effective as doxorubicin in terms of patients' responses to therapy. There was no difference in cardiotoxicity between the two treatment arms. However, in this study, the mortality due to neutropenic sepsis was significantly higher among patients treated with epirubicin.     

Invasive group A streptococcal disease in Taiwan is not associated with the presence of streptococcal pyrogenic exotoxin genes

We reviewed the clinical features of 44 patients with invasive group A streptococcal (GAS) disease who were treated at two teaching hospitals in southern Taiwan from 1991 to 1994. Genes encoding streptococcal pyrogenic exotoxin types A (speA), B (speB), C (speC), and F (speF) and serotypes of M1, M6, and M12 were determined by polymerase chain reaction to target specific sequences in the 44 isolates recovered from these patients and in 28 isolates recovered from upper respiratory sites in 28 additional patients during the study period. The protease activity of these isolates was tested by using the casein plate method. Of the 44 patients with invasive diseases, 25 (57%) had no obvious underlying diseases, and 14 (32%) had preexisting neoplastic diseases or had previously used steroids. Twenty-five patients (57%) presented with cellulitis or necrotizing fasciitis, 24 (55%) had bacteremia, and eight (18%) had streptococcal toxic shock syndrome (STSS). Eight patients (18%) died of invasive GAS disease; seven had STSS, and seven had underlying diseases. All eight patients died within 48 hours after hospitalization. The presence of speA, speC, or speF was not implicated in any particular clinical syndrome in patients with invasive GAS disease. High-level protease activity and the M1 serotype of the isolates were significantly associated with the clinical signs of STSS and with mortality. M1 serotype and protease activity, as well as host immune status, might play significant roles in the pathogenesis of invasive GAS disease in Taiwan.     

Fracture toughness of multilayer pipes

Multilayer pipes composed of various materials improve partially the properties of a pipe system and are frequently used in service. To estimate the lifetime of these pipes the basic fracture parameters have to be measured. In the contribution a new approach to this estimation is presented. Special type of a C-shaped inhomogeneous fracture mechanics specimen machined directly from a pipe has been proposed, numerically analyzed and tested. The corresponding K values are calculated by finite-element method and fracture toughness values of polyethylene pipes material are obtained. __________ Translated from Problemy Prochnosti, No. 1, pp. 146–149, January–February, 2008.     

Cold atmospheric pressure plasma treatment of ready-to-eat meat: inactivation of Listeria innocua and changes in product quality

The application of cold atmospheric pressure plasma for decontamination of a sliced ready-to-eat (RTE) meat product (bresaola) inoculated with Listeria innocua was investigated. Inoculated samples were treated at 15.5, 31, and 62 W for 2–60 s inside sealed linear-low-density-polyethylene bags containing 30% oxygen and 70% argon. Treatments resulted in a reduction of L. innocua ranging from 0.8 ± 0.4 to 1.6 ± 0.5 log cfu/g with no significant effects of time and intensity while multiple treatments at 15.5 and 62 W of 20 s with a 10 min interval increased reduction of L. innocua with increasing number of treatments. Concentrations of thiobarbituric acid reactive substances (TBARS) increased with power, treatments and storage time and were significantly higher than those of control samples after 1 and 14 days of storage at 5 °C. However, the levels were low (from 0.1 to 0.4 mg/kg) and beneath the sensory threshold level. Surface colour changes included loss of redness of ∼40% and 70% after 1 and 14 days of storage, respectively, regardless of plasma treatment. The results indicate that plasma may be applicable in surface decontamination of pre-packed RTE food products. However, oxidation may constitute an issue in some products.     

Hierarchical parameter identification in models of respiratory mechanics

Potential harmful effects of ventilation therapy could be reduced by model-based predictions of the effects of ventilator settings to the patient. To obtain optimal predictions, the model has to be individualized based on patients' data. Given a nonlinear model, the result of parameter identification using iterative numerical methods depends on initial estimates. In this work, a feasible hierarchical identification process is proposed and compared to the commonly implemented direct approach with randomized initial values. The hierarchical approach is exemplarily illustrated by identifying the viscoelastic model (VEM) of respiratory mechanics, whose a priori identifiability was proven. To demonstrate its advantages over the direct approach, two different data sources were employed. First, correctness of the approach was shown with simulation data providing controllable conditions. Second, the clinical potential was evaluated under realistic conditions using clinical data from 13 acute respiratory distress syndrome (ARDS) patients. Simulation data revealed that the success rate of the direct approach exponentially decreases with increasing deviation of the initial estimates while the hierarchical approach always obtained the correct solution. The average computing time using clinical data for the direct approach equals 4.77?s (SD = 1.32) and 2.41?s (SD = 0.01) for the hierarchical approach. These investigations demonstrate that a hierarchical approach may be beneficial with respect to robustness and efficiency using simulated and clinical data.