Analysis of Citrate Content in Bone Using High Performance Liquid Chromatography

Approximately 1.5 wt. % of bone is comprised of citrate molecules bound to the surface of apatite crystals. Furthermore, 80 to 90% of physiological citrate is contained within bone. Recently, interest in citrate has increased due to the potential of a citrate based method for estimation of postmortem intervals, time since death, of skeletal remains. The broad objective of this research was to develop and validate a High-Performance Liquid Chromatography (HPLC) method for quantitative and qualitative analysis of citrate in bone. An appropriate HPLC column and operating conditions for the detection of citrate were selected and subsequently used to analyze standard solutions consisting of sodium citrate dihydrate in water from 0 to 2.5 mM, a physiological range, for linearity, impact of storage time, and intra-day test method precision. After testing on simple matrices, an existing bone processing protocol was optimized by determining the optimal mass of sample, presence of matrix effects, and most suitable bone type to sample (cortical or trabecular). Finally, the HPLC method and optimized processing method were combined to analyze the impact of short term (14 day) storage conditions, intra-bone factors associated with sampling at different anatomical locations, and applicability to a model for the determination of postmortem interval (PMI). The HPLC method developed in this study demonstrated acceptable linearity over a physiological range of standards (0 to 2.5 mM) in all sets of standards analyzed (n =11). Also, the method provided repeatable results under normal operating conditions. Analysis of three bone masses (50, 75, and 100 mg) and different bone types (cortical and trabecular) demonstrated 75 mg, cortical only bone samples were most suitable for analysis. Furthermore, standard addition and recovery tests indicated the presence of matrix effects within the processing protocol of the bones samples. Finally, a total of 36 bone specimens were prepared with the optimized processing protocol and analyzed for citrate. Chromatograms of each sample were analyzed for peak resolution and separation, as well as used to calculate the normalized concentration of citrate (wt. %.). Postmortem intervals were then calculated using a previously published citrate degradation model by Schwarcz et al. in order to assess precision. The mean concentration of citrate in all samples analyzed in this thesis was 1.12 ± 0.6 wt. %, which translated into a mean calculated PMI of 7.93 ± 13.72 years. The results from this thesis demonstrate the need for a better model of PMI estimation, as well as further research on the distribution of citrate in bone

The calculated circular dichroism of polyproline ii in the polarizability approximation

The circular dichroism (CD) spectrum of polyproline II (PPII) has heretofore been moderately well calculated from exciton theory only at the expense of assuming unreasonable chain conformations and accepting a conservative spectrum in the 180–250-nm region (which is not observed). We have incorporated far uv transitions in the polarizability approximation and, together with the Π 2 Π* transition, have calculated the resulting correction to the exciton model. This has been accompanied by a modified assignment of the ΠΠ* transition in PPII, and a simultaneous calculation of the absorption and CD spectra of the Α-helix, Β structure, PPI, and PPII. We obtain good agreement with the observed CD spectrum of PPII in the 180–250-nm region for acceptable chain conformations. In addition, we predict a negative CD into the far uv, in agreement with recent experimental observations. Our calculations also reproduce features of the far uv CD spectrum of the Α-helix, and are in agreement with the CD spectra of the Β chain and PPI. The calculated CD of the unordered polypeptide chain is not significantly influenced by far uv contributions, indicating that our previous calculation is valid for such a system. These results demonstrate the importance of incorporating far uv transitions in order to achieve an adequate theoretical explanation of the CD spectra of polypeptides.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37836/1/360130810_ftp.pd

The Circular Dichroism Spectrum and Structure of Unordered Polypeptides and Proteins

The evidence bearing on our revision of the early assignment of the circular dichroism spectrum of the maximally unordered polypeptide chain is reviewed. Our initial, as well as subsequent, studies are believed to support the proposed revision in assignment. This conclusion need not be modified in the light of recent work on water‐soluble, non‐ionizable polymers.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/101811/1/197400018_ftp.pd

Theoretical calculation of the circular dichroism of unordered polypeptide chains

A calculation has been done of the circular dichroism (CD) spectrum of an unordered polypeptide chain. This has been based on a Boltzmann averaging over a dipeptide conformational CD map. This is shown to be valid by comparing the CD spectra of 28-mer oligopeptides with those generated by summing dipeptide CD spectra. The calculated CD spectrum of an unordered polypeptide chain is found to agree with the assignment proposed by Tiffany and Krimm from experimental studies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37833/1/360110912_ftp.pd

Evaluation of Morphological Plasticity in the Cerebella of Basketball Players with MRI

Cerebellum is a key structure involved in motor learning and coordination. In animal models, motor skill learning increased the volume of molecular layer and the number of synapses on Purkinje cells in the cerebellar cortex. The aim of this study is to investigate whether the analogous change of cerebellar volume occurs in human population who learn specialized motor skills and practice them intensively for a long time. Magnetic resonance image (MRI)-based cerebellar volumetry was performed in basketball players and matched controls with V-works image software. Total brain volume, absolute and relative cerebellar volumes were compared between two groups. There was no significant group difference in the total brain volume, the absolute and the relative cerebellar volume. Thus we could not detect structural change in the cerebellum of this athlete group in the macroscopic level

Photoinduced energy- and electron-transfer from a photoactive coordination cage to bound guests.

In a coordination cage which contains an array of twelve naphthyl chromophores surrounding a central cavity, photoinduced energy or electron-transfer can occur from the chromophore array to the bound guest in supramolecular host/guest complexes

The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana

The plant-specific GRAS/SCL transcription factors play diverse roles in plant development and stress responses. In this study, a poplar SCL gene, PeSCL7, was functionally characterized in Arabidopsis thaliana, especially with regard to its role in abiotic stress resistance. Expression analysis in poplar revealed that PeSCL7 was induced by drought and high salt stresses, but was repressed by gibberellic acid (GA) treatment in leaves. Transient expression of GFP-PeSCL7 in onion epidermal cells revealed that the PeSCL7 protein was localized in the nucleus. Transgenic Arabidopsis plants overexpressing PeSCL7 showed enhanced tolerance to drought and salt treatments. The activity of two stress-responsive enzymes was increased in transgenic seedlings. Taken together, these results suggest that PeSCL7 encodes a member of the stress-responsive GRAS/SCL transcription factors that is potentially useful for engineering drought- and salt-tolerant trees

Molecular analysis of SCARECROW genes expressed in white lupin cluster roots

The Scarecrow (SCR) transcription factor plays a crucial role in root cell radial patterning and is required for maintenance of the quiescent centre and differentiation of the endodermis. In response to phosphorus (P) deficiency, white lupin (Lupinus albus L.) root surface area increases some 50-fold to 70-fold due to the development of cluster (proteoid) roots. Previously it was reported that SCR-like expressed sequence tags (ESTs) were expressed during early cluster root development. Here the cloning of two white lupin SCR genes, LaSCR1 and LaSCR2, is reported. The predicted amino acid sequences of both LaSCR gene products are highly similar to AtSCR and contain C-terminal conserved GRAS family domains. LaSCR1 and LaSCR2 transcript accumulation localized to the endodermis of both normal and cluster roots as shown by in situ hybridization and gene promoter::reporter staining. Transcript analysis as evaluated by quantitative real-time-PCR (qRT-PCR) and RNA gel hybridization indicated that the two LaSCR genes are expressed predominantly in roots. Expression of LaSCR genes was not directly responsive to the P status of the plant but was a function of cluster root development. Suppression of LaSCR1 in transformed roots of lupin and Medicago via RNAi (RNA interference) delivered through Agrobacterium rhizogenes resulted in decreased root numbers, reflecting the potential role of LaSCR1 in maintaining root growth in these species. The results suggest that the functional orthologues of AtSCR have been characterized