Atomic force microscopy investigations of peptide self-assembly

The ability of short peptide fragments to self-assemble in isolation as amyloid and amyloid-like structures has prompted their use as model systems for the study of amyloid formation and recently also for their utilisation as novel nanofibrillar material. The atomic force microscope (AFM) is used here to investigate the self-assembly of two peptide systems and the development of strategies to directly manipulate and control the structures they form. The studies presented in Chapter 2 address the self-assembly of a peptide fragment of the human amylin polypeptide; amylin (20-29). In the opening study we use ex situ AFM imaging to characterise the early stages of amylin (20-29) fibril formation. High-resolution images reveal that following an initial lag phase, fibrils displaying a globular appearance are formed, which over time are replaced by flat ribbon-like fibrils with no periodicity displaying a range of polymorphic structures and assemblies. Following on from these findings, we investigate the influence of solution conditions on amylin (20-29) fibril formation utilising in situ AFM imaging. Altering the pH and electrolyte composition affords a range of morphologies including, truncated and long branched or unbranched flexible fibrils and globular aggregates. Following on from this characterisation chapter, in Chapter 3 a strategy to assemble specifically functionalised fibrillar material from chemically modified amylin (20-29) peptides was investigated. Azide and alkyne moieties were successfully coupled to the amylin (20-29) peptides. Ex situ AFM imaging and Congo red binding confirmed that the additional steric bulk had no detrimental effects on the fibril forming capacity of the peptides. Finally, in Chapter 4 the focus turns to the self-assembly of a dipeptide of phenylalanine which corresponds to the core recognition motif of the beta-amyloid polypeptide. Here, the AFM is used to study the physical properties of the well-ordered, discrete, hollow nanotubes which are formed. Their chemical stability in organic solvents and considerable thermal stability under both dry and wet heating conditions is revealed. Finally, the use of strong magnetic fields to directly control and orientate the diphenylalanine nanotubes was examined by AFM. The results presented throughout this thesis demonstrate the versatility of self-assembling peptides for the generation of fibrillar nanostructures that can be directly modified and controlled to generate novel architectures and functionalised well ordered nanomaterials

Association of endocrine active environmental compounds with body mass index and weight loss following bariatric surgery

IntroductionThe objective of this study was to study associations of a wide range of halogenated biphenyls, dibenzo‐p‐dioxins, dibenzofurans, and diphenylethers with body mass index (BMI) and evaluate changes in their concentration following bariatric surgery.MethodsSubcutaneous fat, visceral fat, and liver tissue samples were collected from 106 patients undergoing Roux‐en‐Y gastric bypass surgery for weight loss or patients who were undergoing abdominal surgery for non‐bariatric reasons. We measured concentrations of an extensive panel of chlorinated and brominated biphenyls, dioxins, and furans, and brominated diphenylethers in the samples. We conducted linear regression to examine associations with BMI, adjusting for age and gender. Changes in concentration for indicator chemicals were evaluated in samples collected following bariatric surgery in a small sub‐population.ResultsAfter adjustments for age and gender and correction for multiple testing, seven ortho‐chlorinated biphenyls, one non‐ortho‐chlorinated biphenyl, four PCDD/F’s and one ortho‐brominated biphenyl were associated with BMI. The strongest associations between BMI and lipid‐adjusted concentrations were seen with PCB‐105 in subcutaneous fat (beta=16.838 P‐val=1.45E‐06) PCB‐126 in visceral fat (beta=15.067 P‐val=7.72E‐06) and PCB‐118 (beta=14.101 P‐val=2.66E‐05) in liver. The concentrations of sum PCBs, chlorinated toxic equivalent quantity (TEQ's) and brominated compounds increased significantly with weight loss in subcutaneous fat in a group of ten individuals resampled up to five years after bariatric surgery and substantial weight loss.ConclusionWe show that selected polychlorinated biphenyls PCBs and structurally related polychlorinated dibenzo‐p‐dioxins dibenzofurans (PCDD/Fs) were associated with BMI. Concentrations of these lipophilic compounds in subcutaneous fat increased following bariatric surgery

Diagnosis of autosomal dominant polycystic kidney disease in utero and in the young infant.

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135563/1/jum198765249.pd

Improved physiology and metabolic flux after Roux-en-Y gastric bypass is associated with temporal changes in the circulating microRNAome: A longitudinal study in humans

BackgroundThe global pandemic of obesity and the metabolic syndrome are leading causes of mortality and morbidity. Bariatric surgery leads to sustained weight loss and improves obesity-associated morbidity including remission of type 2 diabetes. MicroRNAs are small, endogenous RNAs that regulate gene expression post-transcriptionally, controlling most of the human transcriptome and contributing to the regulation of systemic metabolism. This preliminary, longitudinal, repeat sampling study, in which subjects acted as their own control, aimed to assess the temporal effect of bariatric surgery on circulating microRNA expression profiles.MethodsWe used Exiqon’s optimized circulating microRNA panel (comprising 179 validated miRNAs) and miRCURY locked nucleic acid plasma/serum Polymerase Chain Reaction (PCR) to assess circulating microRNA expression. The microRNAome was determined for Roux-en-Y gastric bypass (RYGB) patients examined preoperatively and at 1 month, 3 months, 6 months, 9 months and 12 months postoperatively. Data was analysed using multivariate and univariate statistics.ResultsCompared to the preoperative circulating microRNA expression profile, RYGB altered the circulating microRNAome in a time dependent manner and the expression of 48 circulating microRNAs were significantly different. Importantly, these latter microRNAs are associated with pathways involved in regulation and rescue from metabolic dysfunction and correlated with BMI, the percentage of excess weight loss and fasting blood glucose levels.ConclusionsThe results of this pilot study show that RYGB fundamentally alters microRNA expression in circulation with a time-dependent progressive departure in profile from the preoperative baseline and indicate that microRNAs are potentially novel biomarkers for the benefits of bariatric surgery

Hepatic Abnormalities Associated with Aluminum Loading in Piglets

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141476/1/jpen0293.pd

Aluminum toxicity in childhood

Aluminum intoxication is an iatrogenic disease caused by the use of aluminum compounds for phosphate binding and by the contamination of parenteral fluids. Although organ aluminum deposition was noted as early as 1880 and toxicity was documented in the 1960s, the inability to accurately measure serum and tissue aluminum prevented delineation of its toxic effects until the 1970s. Aluminum toxicity has now been conclusively shown to cause encephalopathy, metabolic bone disease, and microcytic anemia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47831/1/467_2004_Article_BF00869743.pd

C-Terminal Extension of the Yeast Mitochondrial DNA Polymerase Determines the Balance between Synthesis and Degradation

Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo

Pharmacokinetics of orally administered acetaminophen in man

Average and individual sets of plasma concentration-time data for acetaminophen following two oral treatments were simultaneously fitted to the integrated equation describing the two-compartment open model with first-order absorption and lag time. The nonlinear least-squares program NONLIN and an IBM 360/67 digital computer were employed to estimate nine parameters (k A , k B , C A 0 , C B 0 , k 12 , k 21 , k el , and ). When the mean plasma concentrations were weighted according to the inverse of their variances, the parameter estimates more accurately reflected those for individual subjects in the disposition portion of the model. Depending on the relative magnitudes of the disposition rate constants (k 12 , k 21 , and k el ), the one-compartment open model can be used to predict equilibrium-state plasma levels even though the drug is really “two compartment.” Equations are presented which show when the one-compartment approximation is justified. Equations are also presented for calculation of loading doses for multiple dose regimens of any drug obeying the two-compartment open model and the equations are applied to acetaminophen .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45044/1/10928_2005_Article_BF01071309.pd

The Interdomain Linker of AAV-2 Rep68 Is an Integral Part of Its Oligomerization Domain: Role of a Conserved SF3 Helicase Residue in Oligomerization

The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains