Gender-related differences in susceptibility to oxidative stress in healthy middle-aged Serbian adults

Gender-related differences in the association between polymorphism of xenobiotic-metabolising enzymes or non-genetic biomarkers and susceptibility to oxidative stress was assessed in healthy middle-aged Serbian adults, by urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG/creatinine) and total antioxidant status in serum (TAOS). Females were more susceptible to oxidative stress. In both genders, positive predictor of the antioxidative protection was serum triglyceride, while BMI?<25?kg/m² was associated with oxidative stress. Susceptibility to oxidative stress in males was associated with GSTT1*null allele and increased serum iron, but in females, it was decreased serum bilirubin. Early identification of the risk factors could be important in the prevention of oxidative stress-related diseases.     

Efficient transformation ofMicromonospora melanosporea protoplasts byStreptomyces plasmid

An efficient and relatively simple procedure forMicromonospora melanosporea protoplast preparation and transformation is described. Transformation ofM. melanosporea protoplast by theStreptomyces plasmid pIJ702 was optimized by altering parameters affecting the formation, regeneration, and transformation of protoplasts. Improvement of regeneration medium resulted in relatively quick growth of transformants (only 7 days). As a result of these experiments we describe a new transformation method that has routinely yielded 10⁶ transformants/µg plasmid DNA.     

Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm

The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K(D) (binding constant) of 17.6 microM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.     

Patients with solid tumors treated with high-temperature whole body hyperthermia show a redistribution of naive/memory T-cell subtypes

An activation of the immune system might contribute to the therapeutic effect of whole body hyperthermia (WBH) in cancer patients. We explored immune and endocrine responses in patients undergoing high-temperature WBH. Identical parameters were investigated in a separate group of healthy volunteers undergoing physical exercise to rule out effects of sympathetic activation. Lymphocyte subpopulations, lymphocytic expression of a range of adhesion molecules, and serum concentrations of a variety of hormones and cytokines were assessed in cancer patients undergoing high-temperature (60 min at 41.0-41.8 degrees C) WBH (n = 25) and in a separate group of healthy volunteers (n = 10) performing strenuous physical exercise. WBH induced an increase in human growth hormone (hGH), ACTH, and cortisol as well as in TNF-alpha, IL-6, IL-8, and IL-12R. We observed an increase in natural killer (NK) cells and CD56+ NK T cells shortly after initiation of WBH. In contrast, we found a decrease in T cells expressing L-selectin (CD62L) or alpha4beta7 integrin adhesion molecules mediating homing to lymphatic tissues. Accordingly, we observed a decrease in CD45RA+CCR7+ naive and CD45RA-CCR7+ central memory T cells. Numbers of CD45RA-CCR7- memory effector and CD45RA+CCR7 terminally differentiated T cells, on the other hand, remained unchanged. No comparable changes were observed in the group of healthy volunteers. In conclusion, patients with solid tumors treated with WBH show an increase in NK and NK T cells. In a later phase, plasma concentrations of IL-8, hGH, and cortisol increase, correlated with an influx of neutrophils into the peripheral blood. The alterations in T-cell populations suggest that WBH may induce naive and central-memory T cells to enter lymphatic tissue to await antigen exposure and effector T cells to migrate into peripheral tissues to exert their effector function. Although the exercise group may not be an appropriate control to proof the effect of WBH, these changes were not seen in the healthy volunteers performing physical exercise.     

Cross-presentation of HLA class I epitopes from exogenous NY-ESO-1 polypeptides by nonprofessional APCs

NY-ESO-1, a germ cell Ag often detected in tumor tissues, frequently elicits Ab and CD8(+) T cell responses in cancer patients. Overlapping long peptides spanning the NY-ESO-1 sequence have been used to map HLA class I-restricted epitopes recognized by NY-ESO-1-specific CD8(+) T lymphocytes. To address the antigenicity of long peptides, we analyzed two synthetic 30-mer peptides from NY-ESO-1, polypeptides 80-109 and 145-174, for their capacity to be processed by APCs and to stimulate CD8(+) T cells. By incubating APCs with polypeptides at different temperatures or in the presence of protease inhibitors, we found that NY-ESO-1 polypeptides were rapidly internalized by B cells, T2 cells, or PBLs and submitted to cellular proteolytic action to yield nonamer epitopes presented by HLA class I. Polypeptides were also immunogenic in vitro and stimulated the expansion of CD8(+) T cells against naturally processed NY-ESO-1 epitopes in the context of three different HLA class I alleles. Polypeptides can thus serve as exogenous Ags that are cross-presented on HLA class I without requiring the action of professional APCs. These findings support innovative vaccination strategies using NY-ESO-1 polypeptides that would circumvent current limitations of HLA class I peptide vaccination, i.e., HLA eligibility criteria and knowledge of epitope, while allowing for facilitated immunogenicity in the presence of helper epitopes.     

41.8 degrees C whole body hyperthermia as an adjunct to chemotherapy induces prolonged T cell activation in patients with various malignant diseases

Whole body hyperthermia (WBH) has been used as an adjunct to radio-/chemotherapy in patients with various malignant diseases. Although clear evidence is still missing, it has been hypothesized that an activation of the immune system might contribute to the therapeutic effect of WBH. To examine whether a treatment with 60-minute 41.8 degrees C WBH as an adjunct to chemotherapy (WBH-CT) induces an activation of T cells, blood samples were collected at numerous time points before and up to 48 h post-treatment. The aim of this study was to examine the effect of WBH-CT on the expression of a broad range of activation markers on peripheral blood lymphocytes (PBL), on serum cytokines and intracellular cytokine levels in T cells, and the capacity of these cells to proliferate. Immediately after 41.8 degrees C WBH-CT treatment, a drastic increase in peripheral natural killer (NK) cells ( P<0.05) and CD56+ cytotoxic T lymphocytes (CTL; P<0.01) in the patients' peripheral blood was observed. At 5 h post-treatment, the percentages of both effector cell types had returned to baseline levels. This transient phenomenon was accompanied by a short period of reduced T cell activity, indicated by diminished serum levels of soluble interleukin-2 receptors (sIL-2R) at 3 h post-WBH-CT ( P<0.05) and decreased lymphocytic proliferation at the same point in time. This first phase was followed by a marked but short-lived increase in the patients' serum levels of interleukin-6 (IL-6; P<0.01) during the first 5 h following treatment, with a subsequent decrease to baseline levels at 24 h and significantly increased serum levels of tumor necrosis factor-alpha (TNF-alpha) at 0 h ( P<0.01), 3 h ( P<0.05), 5 h ( P<0.05) and 24 h ( P<0.01) post-WBH-CT. The third phase of the immunological consequences of WBH-CT consisted of an increase in the percentage of peripheral cytotoxic T lymphocytes (CTL) expressing CD56, reaching a maximum at 48 h post-WBH ( P<0.01). Furthermore, the percentage of CD4+ T cells expressing the T cell activation marker CD69 increased nearly two-fold over time, reaching its maximum at 48 h ( P<0.05). As an additional marker for T cell activation, serum levels of sIL-2R increased markedly ( P<0.01), reaching maximum levels at the same point in time. Elevated intracellular concentrations of interferon-gamma (IFN-gamma) and/or TNF-alpha in CD8+ T cells were found in 4 out of 5 patients at 24 h post-WBH-CT. Since similar changes were not observed in patients receiving chemotherapy alone, this is the first study to provide evidence for prolonged WBH-CT-induced activation of human T cells.     

Size scaling of mutation avalanches in a model for protein evolution

The tunably rugged NK-model is used to study avalanche-like events that occur when environmental change causes fitness optima to disappear. The probability of an event with Delta substitutions scales as exp(-c Delta) for smooth landscapes, and as exp(-c Delta(2)) for rugged landscapes. Increasing the ruggedness leads to two competing effects: (1) more possible routes by which single mutations can increase the fitness, which dominates at low ruggedness and acts to increase Delta; and (2) a higher density of fitness optima, which dominates at high ruggedness and acts to decrease Delta. Due to these competing effects, the largest average values of Delta occur at intermediate ruggedness. The effects of system size on the avalanche events are examined, and average values of Delta increase logarithmically with system size. The variance to mean ratios for the number of substitutions per unit time are consistent with experimental results for protein evolution.     

Design and synthesis of potent, orally active, inhibitors of carboxypeptidase U (TAFIa)

A series of 3-mercapto-propionic acid derivatives that function as reversible inhibitors of carboxypeptidase U have been prepared. We present a successful design strategy using cyclic, low basicity guanidine mimetics resulting in potent, selective and bioavailable inhibitors of carboxypeptidase U (TAFIa).     

The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3

Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min^?1 g cell dry weight^?1 resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite (S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10–23 %). The rate of benzo[b]thiophene and 2-methylbenzo[b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group.     

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28''s SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.