Western diet, but not high fat diet, causes derangements of fatty acid metabolism and contractile dysfunction in the heart of Wistar rats.

Obesity and diabetes are associated with increased fatty acid availability in excess of muscle fatty acid oxidation capacity. This mismatch is implicated in the pathogenesis of cardiac contractile dysfunction and also in the development of skeletal-muscle insulin resistance. We tested the hypothesis that 'Western' and high fat diets differentially cause maladaptation of cardiac- and skeletal-muscle fatty acid oxidation, resulting in cardiac contractile dysfunction. Wistar rats were fed on low fat, 'Western' or high fat (10, 45 or 60% calories from fat respectively) diet for acute (1 day to 1 week), short (4-8 weeks), intermediate (16-24 weeks) or long (32-48 weeks) term. Oleate oxidation in heart muscle ex vivo increased with high fat diet at all time points investigated. In contrast, cardiac oleate oxidation increased with Western diet in the acute, short and intermediate term, but not in the long term. Consistent with fatty acid oxidation maladaptation, cardiac power decreased with long-term Western diet only. In contrast, soleus muscle oleate oxidation (ex vivo) increased only in the acute and short term with either Western or high fat feeding. Fatty acid-responsive genes, including PDHK4 (pyruvate dehydrogenase kinase 4) and CTE1 (cytosolic thioesterase 1), increased in heart and soleus muscle to a greater extent with feeding a high fat diet compared with a Western diet. In conclusion, we implicate inadequate induction of a cassette of fatty acid-responsive genes, and impaired activation of fatty acid oxidation, in the development of cardiac dysfunction with Western diet.

NOVEL AMINO ACID TRANSPORTER-TARGETED RADIOTRACERS FOR BREAST CANCER IMAGING

Breast cancer is the most common malignancy among women in the world. Its 5-year survival rate ranges from 23.4% in patients with stage IV to 98% in stage I disease, highlighting the importance of early detection and diagnosis. 18F-2-Fluoro-2-deoxy-glucose (18F-FDG), using positron emission tomography (PET), is the most common functional imaging tool for breast cancer diagnosis currently. Unfortunately, 18F-FDG-PET has several limitations such as poorly differentiating tumor tissues from inflammatory and normal brain tissues. Therefore, 18F-labeled amino acid-based radiotracers have been reported as an alternative, which is based on the fact that tumor cells uptake and consume more amino acids to sustain their uncontrolled growth. Among those radiotracers, 18F-labeled tyrosine and its derivatives have shown high tumor uptake and great ability to differentiate tumor tissue from inflammatory sites in brain tumors and squamous cell carcinoma. They enter the tumor cells via L-type amino acid transporters (LAT), which were reported to be highly expressed in many cancer cell lines and correlate positively with tumor growth. Nevertheless, the low radiosynthesis yield and demand of an on-site cyclotron limit the use of 18F-labeled tyrosine analogues. In this study, four Technetium-99m (99mTc) labeled tyrosine/ AMT (α-methyl tyrosine)-based radiotracers were successfully synthesized and evaluated for their potentials in breast cancer imaging. In order to radiolabel tyrosine and AMT, the chelators N,N’-ethylene-di-L-cysteine (EC) and 1,4,8,11-tetra-azacyclotetradecane (N4 cyclam) were selected to coordinate 99mTc. These chelators have been reported to provide stable chelation ability with 99mTc. By using the chelator technology, the same target ligand could be labeled with different radioisotopes for various imaging modalities for tumor diagnosis, or for internal radionuclide therapy in future. Based on the in vitro and in vivo evaluation using the rat mammary tumor models, 99mTc-EC-AMT is considered as the most suitable radiotracer for breast cancer imaging overall, however, 99mTc-EC-Tyrosine will be more preferred for differential diagnosis of tumor from inflammation.

STUDIES ON THE PATTERNS OF NUCLEOTIDE AND AMINO ACID SUBSTITUTION (ELECTROPHORESIS, MUTATION RATE, SELECTION, BASE, COMPOSITION)

Theoretical and empirical studies were conducted on the pattern of nucleotide and amino acid substitution in evolution, taking into account the effects of mutation at the nucleotide level and purifying selection at the amino acid level. A theoretical model for predicting the evolutionary change in electrophoretic mobility of a protein was also developed by using information on the pattern of amino acid substitution. The specific problems studied and the main results obtained are as follows: (1) Estimation of the pattern of nucleotide substitution in DNA nuclear genomes. The pattern of point mutations and nucleotide substitutions among the four different nucleotides are inferred from the evolutionary changes of pseudogenes and functional genes, respectively. Both patterns are non-random, the rate of change varying considerably with nucleotide pair, and that in both cases transitions occur somewhat more frequently than transversions. In protein evolution, substitution occurs more often between amino acids with similar physico-chemical properties than between dissimilar amino acids. (2) Estimation of the pattern of nucleotide substitution in RNA genomes. The majority of mutations in retroviruses accumulate at the reverse transcription stage. Selection at the amino acid level is very weak, and almost non-existent between synonymous codons. The pattern of mutation is very different from that in DNA genomes. Nevertheless, the pattern of purifying selection at the amino acid level is similar to that in DNA genomes, although selection intensity is much weaker. (3) Evaluation of the determinants of molecular evolutionary rates in protein-coding genes. Based on rates of nucleotide substitution for mammalian genes, the rate of amino acid substitution of a protein is determined by its amino acid composition. The content of glycine is shown to correlate strongly and negatively with the rate of substitution. Empirical formulae, called indices of mutability, are developed in order to predict the rate of molecular evolution of a protein from data on its amino acid sequence. (4) Studies on the evolutionary patterns of electrophoretic mobility of proteins. A theoretical model was constructed that predicts the electric charge of a protein at any given pH and its isoelectric point from data on its primary and quaternary structures. Using this model, the evolutionary change in electrophoretic mobilities of different proteins and the expected amount of electrophoretically hidden genetic variation were studied. In the absence of selection for the pI value, proteins will on the average evolve toward a mildly basic pI. (Abstract shortened with permission of author.) ^

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

Acetate metabolism and the control of environmental pH in Candida albicans and Saccharomyces cerevisiae

Candida albicans is the most common opportunistic fungal pathogen of humans. The balance between commensal and pathogenic C. albicans is maintained largely by phagocytes of the innate immune system. Analysis of transcriptional changes after macrophage phagocytosis indicates the C. albicans response is broadly similar to starvation, including up-regulation of alternate carbon metabolism. Systems known and suspected to be part of acetate/acetyl-CoA metabolism were also up-regulated, importantly the ACH and ACS genes, which manage acetate/acetyl-CoA interconversion, and the nine-member ATO gene family, thought to participate in transmembrane acetate transport and also linked to the process of environmental alkalinization. ^ Studies into the roles of Ach, Acs1 and Acs2 function in alternate carbon metabolism revealed a substantial role for Acs2 and lesser, but distinct roles, for Ach and Acs1. Deletion mutants were made in C. albicans and were phenotypically evaluated both in vitro and in vivo. Loss of Ach function resulted in mild growth defects on ethanol and acetate and no significant attenuation in virulence in a disseminated mouse model of infection. While loss of Acs1 did not produce any significant phenotypes, loss of Acs2 greatly impaired growth on multiple carbon sources, including glucose, ethanol and acetate. We also concluded that ACS1 and ACS2 likely comprise an essential gene pair. Expression analyses indicated that ACS2 is the predominant form under most growth conditions. ^ ATO gene function had been linked to the process of environmental alkalinization, an ammonium-mediated phenomenon described here first in C. albicans. During growth in glucose-poor, amino acid-rich conditions C. albicans can rapidly change its extracellular pH. This process was glucose-repressible and was accompanied by hyphal formation and changes in colony morphology. We showed that introduction of the ATO1G53D point mutant to C. albicans blocked alkalinization, as did over-expression of C. albicans ATO2, the only C. albicans ATO gene to lack the conserved N-terminal domain. A screen for alkalinization-deficient mutants revealed that ACH1 is essential for alkalinization. However, addition of acetate to the media restored alkalinization to the ach1 mutant. We proposed a model of ATO function in which Atos regulated the cellular co-export of ammonium and acetate. ^

Development of combined micro -preparation, separation, and mass spectrometry methods and application in the microdialysis study of neuropeptide metabolism

Two sets of mass spectrometry-based methods were developed specifically for the in vivo study of extracellular neuropeptide biochemistry. First, an integrated micro-concentration/desalting/matrix-addition device was constructed for matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) to achieve attomole sensitivity for microdialysis samples. Second, capillary electrophoresis (CE) was incorporated into the above micro-liquid chromatography (LC) and MALDI MS system to provide two-dimensional separation and identification (i.e. electrophoretic mobility and molecular mass) for the analysis of complex mixtures. The latter technique includes two parts of instrumentation: (1) the coupling of a preconcentration LC column to the inlet of a CE capillary, and (2) the utilization of a matrix-precoated membrane target for continuous CE effluent deposition and for automatic MALDI MS analysis (imaging) of the CE track.^ Initial in vivo data reveals a carboxypeptidase A (CPA) activity in rat brain involved in extracellular neurotensin metabolism. Benzylsuccinic acid, a CPA inhibitor, inhibited neurotensin metabolite NT1-12 formation by 70%, while inhibitors of other major extracellular peptide metabolizing enzymes increased NT1-12 formation. CPA activity has not been observed in previous in vitro experiments. Next, the validity of the methodology was demonstrated in the detection and structural elucidation of an endogenous neuropeptide, (L)VV-hemorphin-7, in rat brain upon ATP stimulation. Finally, the combined micro-LC/CE/MALDI MS was used in the in vivo metabolic study of peptide E, a mu-selective opioid peptide with 25 amino acid residues. Profiles of 88 metabolites were obtained, their identity being determined by their mass-to-charge ratio and electrophoretic mobility. The results indicate that there are several primary cleavage sites in vivo for peptide E in the release of its enkephalin-containing fragments. ^