A study of methods of testing and sampling technique in the determination of fat content of ground meat

Call number: LD2668 .T4 1952 K42Master of Scienc

Sphingosine protects aging hearts from ischemia/reperfusion injury: Superiority to sphingosine 1-phosphate and ischemic pre- and post-conditioning

Aging hearts are known to have diminished capacity to be protected against reoxygenation ischemia/reperfusion (IR) injury provided by various cardioprotective regimens. In search of a more successful regimen, we have studied the response of aged hearts to preconditioning (PC) and postconditioning (POST) elicited by sphingosine or sphingosine 1-phosphate treatment

SAR by MS for Functional Genomics (Structure-Activity Relation by Mass Spectrometry)

Large-scale functional genomics will require fast, high-throughput experimental techniques, coupled with sophisticated computer algorithms for data analysis and experiment planning. In this paper, we introduce a combined experimental-computational protocol called Structure-Activity Relation by Mass Spectrometry (SAR by MS), which can be used to elucidate the function of protein-DNA or protein-protein complexes. We present algorithms for SAR by MS and analyze their complexity. Carefully-designed Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI TOF) and Electrospray Ionization (ESI) assays require only femtomolar samples, take only microseconds per spectrum to record, enjoy a resolution of up to one dalton in $10^6$, and (in the case of MALDI) can operate on protein complexes up to a megadalton in mass. Hence, the technique is attractive for high-throughput functional genomics. In SAR by MS, selected residues or nucleosides are 2H-, 13C-, and/or 15N-labeled. Second, the complex is crosslinked. Third, the complex is cleaved with proteases and/or endonucleases. Depending on the binding mode, some cleavage sites will be shielded by the crosslinking. Finally, a mass spectrum of the resulting fragments is obtained and analyzed. The last step is the Data Analysis phase, in which the mass signatures are interpreted to obtain constraints on the functional binding mode. Experiment Planning entails deciding what labeling strategy and cleaving agents to employ, so as to minimize mass degeneracy and spectral overlap, in order that the constraints derived in data analysis yield a small number of binding hypotheses. A number of combinatorial and algorithmic questions arise in deriving algorithms for both Experiment Planning and Data Analysis. We explore the complexity of these problems, obtaining upper and lower bounds. Experimental results are reported from an implementation of our algorithms

A Sphingosine Kinase Form 2 Knockout Sensitizes Mouse Myocardium to Ischemia/Reoxygenation Injury and Diminishes Responsiveness to Ischemic Preconditioning

Sphingosine kinase (SphK) exhibits two isoforms, SphK1 and SphK2. Both forms catalyze the synthesis of sphingosine 1-phosphate (S1P), a sphingolipid involved in ischemic preconditioning (IPC). Since the ratio of SphK1 : SphK2 changes dramatically with aging, it is important to assess the role of SphK2 in IR injury and IPC. Langendorff mouse hearts were subjected to IR (30 min equilibration, 50 min global ischemia, and 40 min reperfusion). IPC consisted of 2 min of ischemia and 2 min of reperfusion for two cycles. At baseline, there were no differences in left ventricular developed pressure (LVDP), ± dP/dtmax, and heart rate between SphK2 null (KO) and wild-type (WT) hearts. In KO hearts, SphK2 activity was undetectable, and SphK1 activity was unchanged compared to WT. Total SphK activity was reduced by 53%. SphK2 KO hearts subjected to IR exhibited significantly more cardiac damage (37 ± 1% infarct size) compared with WT (28 ± 1% infarct size); postischemic recovery of LVDP was lower in KO hearts. IPC exerted cardioprotection in WT hearts. The protective effect of IPC against IR was diminished in KO hearts which had much higher infarction sizes (35 ± 2%) compared to the IPC/IR group in control hearts (12 ± 1%). Western analysis revealed that KO hearts had substantial levels of phosphorylated p38 which could predispose the heart to IR injury. Thus, deletion of the SphK2 gene sensitizes the myocardium to IR injury and diminishes the protective effect of IPC

The NOESY Jigsaw: Automated Protein Secondary Structure and Main-Chain Assignment from Sparse, Unassigned NMR Data

High-throughput, data-directed computational protocols for Structural Genomics (or Proteomics) are required in order to evaluate the protein products of genes for structure and function at rates comparable to current gene-sequencing technology. This paper presents the Jigsaw algorithm, a novel high-throughput, automated approach to protein structure characterization with nuclear magnetic resonance (NMR). Jigsaw consists of two main components: (1) graph-based secondary structure pattern identification in unassigned heteronuclear NMR data, and (2) assignment of spectral peaks by probabilistic alignment of identified secondary structure elements against the primary sequence. Jigsaw\u27s deferment of assignment until after secondary structure identification differs greatly from traditional approaches, which begin by correlating peaks among dozens of experiments. By deferring assignment, Jigsaw not only eliminates this bottleneck, it also allows the number of experiments to be reduced from dozens to four, none of which requires 13C-labeled protein. This in turn dramatically reduces the amount and expense of wet lab molecular biology for protein expression and purification, as well as the total spectrometer time to collect data. Our results for three test proteins demonstrate that we are able to identify and align approximately 80 percent of alpha-helical and 60 percent of beta-sheet structure. Jigsaw is extremely fast, running in minutes on a Pentium-class Linux workstation. This approach yields quick and reasonably accurate (as opposed to the traditional slow and extremely accurate) structure calculations, utilizing a suite of graph analysis algorithms to compensate for the data sparseness. Jigsaw could be used for quick structural assays to speed data to the biologist early in the process of investigation, and could in principle be applied in an automation-like fashion to a large fraction of the proteome