Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein

The TcTLE peptide (TLEEFSAKL) is a CD8⁺ T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8⁺ T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8⁺ T cells in HLA-A2⁺ chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8⁺ T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope.     

Meiofaunal responses to nutrient additions in a Mediterranean stream

1. The effects of a moderate addition of nutrients (twofold N and threefold P) were examined during a 2‐year period to determine the response to nutrient addition in a meiofaunal community inhabiting sandy patches in a Mediterranean stream. 2. The pattern of meiofaunal assemblages exhibits a high degree of intra‐ and interannual variability. This pattern alternates between periods of hydrological stability and disturbances, such as floods and droughts, which is a characteristic of Mediterranean systems. 3. A before–after–control–impact (BACI) design was used to determine the outcome of the addition by comparing an upstream non‐enriched reach with an enriched downstream reach. Analysis of the study data by means of a nonparametric permutational procedure (permanova ) showed that fertilisation had a significant effect. Density and biomass values increased in the most abundant meiofaunal groups, including microcrustaceans, oligochaetes and chironomids. Microcrustaceans were the dominant group in the permanent meiofauna. 4. We also examined differences in microcrustacean secondary production in both reaches. Ostracods and cyclopoid copepods increased their secondary production in the impacted reach as a result of the nutrient addition. 5. Our study demonstrated that moderate nutrient enrichment can affect the biomass and production of stream meiofauna, but it is still unclear whether this effect was because of autotrophic or heterotrophic pathways.     

Existence of a Novel Hemagglutinin Having No Protease Activity in Vibrio mimicus

The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease. A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V. mimicus. This HA (V. mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep. Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA. The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease. These results provide evidence for the existence of novel HA other than HA/protease in V. mimicus.     

Multi-Targeting Tacrine Conjugates with Cholinesterase and Amyloid-Beta Inhibitory Activities: New Anti-Alzheimer's Agents

Alzheimer's disease (AD) is a severe age dependent and chronic problem with no cure so far. The available treatments are temporary, acting over short period of time. The main pathological hallmark of the disease includes cholinergic dysfunction, oxidative stress, accumulation of Aβ fibrils and tau tangles. In context with the multi-factorial nature of this disease, two different series of molecules were developed to hit the multifactorial disease targets. Mainly, the molecules were designed to inhibit the AChE and aggregation of Aβ, and also oxidative damage. Two novel series of TAC-fenbufen/menbutone conjugated molecules were designed, synthesized and bio-assayed. All compounds showed inhibition capacity towards AChE, Aβ aggregation and moderate to good radical scavenging capacity. Particularly, five TAC-menbutone molecules showed improved AChE and Aβ aggregation inhibition capacity compared to TAC-fenbufen conjugated molecules. Overall, these novel series of molecules may be potential drug lead molecules in the treatment of AD.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays

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Monoamine oxidase-A is an important source of oxidative stress and promotes cardiac dysfunction,apoptosis, and fibrosis in diabetic cardiomyopathy

Oxidative stress is closely associated with the pathophysiology of diabetic cardiomyopathy (DCM). The mitochondrial flavoenzyme monoamine oxidase A (MAO-A) is an important source of oxidative stress in the myocardium. We sought to determine whether MAO-A plays a major role in modulating DCM. Diabetes was induced in Wistar rats by single intraperitoneal injection of streptozotocin (STZ). To investigate the role of MAO-A in the development of pathophysiological features of DCM, hyperglycemic and age-matched control rats were treated with or without the MAO-A-specific inhibitor clorgyline (CLG) at 1 mg/kg/day for 8 weeks. Diabetes upregulated MAO-A activity; elevated markers of oxidative stress such as cardiac lipid peroxidation, superoxide dismutase activity, and UCP3 protein expression; enhanced apoptotic cell death; and increased fibrosis. All these parameters were significantly attenuated by CLG treatment. In addition, treatment with CLG substantially prevented diabetes-induced cardiac contractile dysfunction as evidenced by decreased QRS, QT, and corrected QT intervals, measured by ECG, and LV systolic and LV end-diastolic pressure measured by microtip pressure transducer. These beneficial effects of CLG were seen despite the persistent hyperglycemic and hyperlipidemic environments in STZ-induced experimental diabetes. In summary, this study provides strong evidence that MAO-A is an important source of oxidative stress in the heart and that MAO-A-derived reactive oxygen species contribute to DCM.     

Identification of amino compounds synthesized and translocated in symbiotic Parasponia

We investigated the synthesis and translocation of amino compounds in Parasponia, a genus of the Ulmaceae that represents the only non-legumes known to form a root nodule symbiosis with rhizohia. In the xylem sap of P. andersonii we identified asparagine. aspartate. glutamine, glutamated significant quantities of a non-protein amino acid. 4-methylglutamte(2-amino-4-methylpentanedioic acid). This identification was confirmed by two methods, capillary gas chromatography (GC) electron ionization (El) mass spectrometry (MS) and reverse phase high pressure liquid chromatography (HPLC) analysis of derivatized compounds. In leaf, root and nodule samples from P. andersonii and P. parviflora we also identified the related compounds 4-methyleneglutamate and 4-methyleneglulamine. Using ¹⁵N₂ labelling and GC-Ms analysis of root nodule extracts we followed N₂ fixation and ammonia assimilation in P. andersonii root nodules and observed Label initially in glutamine and subsequently in glutamate, suggesting operation of the glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) pathway. Importantly, we observed the incorporation of significant quantities of ¹⁵N into 4-methylglutamate in nodules, demonstrating the de nova synthesis of this non protein amino acid and suggesting a role in the translation of N in symbioticParasponia.