Alterations in the red blood cell membrane proteome in alzheimer's subjects reflect disease-related changes and provide insight into altered cell morphology

Background  

Our earlier studies have shown that red blood cell (RBC) morphology in Alzheimer's disease (AD) subjects was altered (> 15% of the RBCs were elongated as compared to 5.9% in normal controls (p < 0.0001)). These results suggested alterations in the RBC membrane architecture in AD subjects, possibly due to RBC-β-amyloid interactions and/or changes in the expression of membrane proteins. We hypothesized that the observed changes could be due to changes in the level of the protein components of the cytoskeleton and those linked to the RBC membrane. To examine this, we performed a proteomic analysis of RBC membrane proteins of AD subjects, and their age-matched controls using one pool of samples from each group, following their separation by SDS-PAGE, in-gel Tryptic digestion, LC-MS-MS of peptides generated, and a label-free approach of semi-quantitative analysis of their relative MS spectral intensities.     

Characterization of high temperature induced stress impairments in thylakoids of rice seedlings.

Exposure of isolated thylakoids or intact plants to elevated temperature is known to inhibit photosynthesis at multiple sites. We have investigated the effect of elevated temperature (40 degrees C) for 24 hr in dark on rice seedlings to characterize the extent of damage by in vivo heat stress on photofunctions of photosystem II (PSII). Chl a fluorescence transient analysis in the intact rice leaves indicated a loss in PSII photochemistry (Fv) and an associated loss in the number of functional PSII units. Thylakoids isolated from rice seedlings exposed to mild heat stress exhibited >50% reduction in PSII catalyzed oxygen evolution activity compared to the corresponding control thylakoids. The ability of thylakoid membranes from heat exposed seedlings to photooxidize artificial PSII electron donor, DPC, subsequent to washing the thylakoids with alkaline Tris or NH2OH was also reduced by approximately 40% compared to control Tris or NH2OH washed thylakoids. This clearly indicated that besides the disruption of oxygen evolving complex (OEC) by 40 degrees C heat exposure for 24 hr, the PSII reaction centers were impaired by in vivo heat stress. The analysis of Mn and manganese stabilizing protein (MSP) contents showed no breakdown of 33 kDa extrinsic MSP and only a marginal loss in Mn. Thus, we suggest that the extent of heat induced loss of OEC must be due to disorganization of the OEC complex by in vivo heat stress. Studies with inhibitors like DCMU and atrazine clearly indicated that in vivo heat stress altered the acceptor side significantly. [14C] Atrazine binding studies clearly demonstrated that there is a significant alteration in the QB binding site on D1 as well as altered QA to QB equilibrium. Thus, our results show that the loss in PSII photochemistry by in vivo heat exposure not only alters the donor side but significantly alters the acceptor side of PSII.     

Mechanism of formation of amyloid protofibrils of barstar from soluble oligomers: evidence for multiple steps and lateral association coupled to conformational conversion

Understanding the heterogeneity of the soluble oligomers and protofibrillar structures that form initially during the process of amyloid fibril formation is a critical aspect of elucidating the mechanism of amyloid fibril formation by proteins. The small protein barstar offers itself as a good model protein for understanding this aspect of amyloid fibril formation, because it forms a stable soluble oligomer, the A form, at low pH, which can transform into protofibrils. The mechanism of formation of protofibrils from soluble oligomer has been studied by multiple structural probes, including binding to the fluorescent dye thioflavin T, circular dichroism and dynamic light scattering, and at different temperatures and different protein concentrations. The kinetics of the increase in any probe signal are single exponential, and the rate measured depends on the structural probe used to monitor the reaction. Fastest is the rate of increase in the mean hydrodynamic radius, which grows from a value of 6 nm for the A form to 20 nm for the protofibril. Slower is the rate of increase in thioflavin T binding capacity, and slowest is the rate of increase in circular dichroism at 216 nm, which occurs at about the same rate as that of the increase in light scattering intensity. The dynamic light scattering measurements suggest that the A form transforms completely into larger size aggregates at an early stage during the aggregation process. It appears that structural changes within the aggregates occur at the late stages of assembly into protofibrils. For all probes, and at all temperatures, no initial lag phase in protofibril growth is observed for protein concentrations in the range of 1 microM to 50 microM. The absence of a lag phase in the increase of any probe signal suggests that aggregation of the A form to protofibrils is not nucleation dependent. In addition, the absence of a lag phase in the increase of light scattering intensity, which changes the slowest, suggests that protofibril formation occurs through more than one pathway. The rate of aggregation increases with increasing protein concentration, but saturates at high concentrations. An analysis of the dependence of the apparent rates of protofibril formation, determined by the four structural probes, indicates that the slowest step during protofibil formation is lateral association of linear aggregates. Conformational conversion occurs concurrently with lateral association, and does so in two steps leading to the creation of thioflavin T binding sites and then to an increase in beta-sheet structure. Overall, the study indicates that growth during protofibril formation occurs step-wise through progressively larger and larger aggregates, via multiple pathways, and finally through lateral association of critical aggregates.     

Structural and functional studies on Ribonuclease S, retro S and retro-inverso S peptides

Ribonuclease S peptide and S protein offer a unique complementation system to understand the finer features of molecular recognition. In the present study the S peptide (1-16), and its retro and retro-inverso analogs have been analyzed for their structural and biological attributes. RPHPLC, CD, and NMR analyses have revealed that the physicochemical and conformational properties of the S peptide are distinct from those of its retro and retro-inverso analogs. On the functional side, while the S peptide complemented the S protein to give RNase activity, was recognized by anti-S peptide antibodies and induced T cell proliferation, neither the retro nor the retro-inverso S peptides could do so.     

In vivo humoral and cellular reactions, and fate of injected bacteria Aeromonas hydrophila in freshwater prawn Macrobrachium rosenbergii

Live non-opsonized and opsonized Aeromonas hydrophila were injected into juveniles of freshwater prawn Macrobrachium rosenbergii to study the cells involved in phagocytosis, distribution of bacteria, cellular reactions and clearance of both forms of bacteria from the system. The bacteria were rapidly distributed to various tissues viz., gills, heart, hepatopancreas within 1h, and the tissues revealed haemocytic nodule formation after 3 h of injection. There was rapid clearance of both the forms of bacteria from the circulation. However, clearance efficiency was significantly (P < 0.05) faster in the case of opsonized bacteria at 12 h after injection. Similarly, the nodule formation, that was prominent in cardiac musculature, was rapidly eliminated from the tissues of the group injected with opsonized bacteria as compared to non-opsonized bacteria injected group, thus confirming the existence of opsonic factors in haemolymph of this prawn. In another experiment, various dose levels of bacteria were injected intramuscularly into prawns and haemolymph was collected after 1, 6, 24, 72 h and 7 days of injection to study various immune parameters. Although, no major alterations in the total and differential haemocyte counts were observed in bacteria injected prawns compared to control, there was a significant decline in phenoloxidase activity in the highest dose bacteria injected group at the earlier phase and a rise in agglutinin levels at the later phase of the experimental period in the higher dose groups.     

Soil microorganisms regulate extracellular enzyme production to maximize their growth rate

Biogeochemistry - Soil carbon cycling and ecosystem functioning can strongly depend on how microbial communities regulate their metabolism and adapt to changing environmental conditions to improve...     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.