Measurement of the vascular input function in mice for DCE-MRI

DCE-MRI is an important technique in the study of small animal cancer models because its sensitivity to vascular changes opens the possibility of quantitative assessment of early therapeutic response. However, extraction of physiologically descriptive parameters from DCE-MRI data relies upon measurement of the vascular input function (VIF), which represents the contrast agent concentration time course in the blood plasma. This is difficult in small animal models due to artifacts associated with partial volume, inflow enhancement, and the limited temporal resolution achievable with MR imaging. In this work, the development of a suite of techniques for high temporal resolution, artifact resistant measurement of the VIF in mice is described. One obstacle in VIF measurement is inflow enhancement, which decreases the sensitivity of the MR signal to the presence of contrast agent. Because the traditional techniques used to suppress inflow enhancement degrade the achievable spatiotemporal resolution of the pulse sequence, improvements can be achieved by reducing the time required for the suppression. Thus, a novel RF pulse which provides spatial presaturation contemporaneously with the RF excitation was implemented and evaluated. This maximizes the achievable temporal resolution by removing the additional RF and gradient pulses typically required for suppression of inflow enhancement. A second challenge is achieving the temporal resolution required for accurate characterization of the VIF, which exceeds what can be achieved with conventional imaging techniques while maintaining adequate spatial resolution and tumor coverage. Thus, an anatomically constrained reconstruction strategy was developed that allows for sampling of the VIF at extremely high acceleration factors, permitting capture of the initial pass of the contrast agent in mice. Simulation, phantom, and in vivo validation of all components were performed. Finally, the two components were used to perform VIF measurement in the murine heart. An in vivo study of the VIF reproducibility was performed, and an improvement in the measured injection-to-injection variation was observed. This will lead to improvements in the reliability of quantitative DCE-MRI measurements and increase their sensitivity.

Intracellular chemical messengers and neuronal function: Two examples

Two distinct classes of neurons have been examined in the nervous system of Aplysia. The membrane properties of these neurons are regulated by intracellular signalling molecules in both a short-term and a long-term fashion.^ The role of the phosphatidylinositol cycle in the control of neuronal properties was studied in a class of bursting pacemaker cells, the left upper-quadrant bursting neurons (cells L2, L3, L4, and L6) of the abdominal ganglion of Aplysia. These cells display a regular burst-firing pattern that is controlled by cyclic changes of intracellular Ca$\sp{2+}$ that occur during the bursting rhythm. The characteristic bursting pattern of these neurons occurs within a range of membrane potentials ($-35$ to $-50$ mV) called the pacemaker range. Intracellular pressure injection of inositol 1,4,5-trisphosphate (IP$\sb3$) altered the bursting rhythm of the bursting cells. Injection of IP$\sb3$ induced a brief depolarization that was followed by a long-lasting (2-15 min) hyperpolarization. When cells were voltage-clamped at potentials within the pacemaker range, injection of IP$\sb3$ generally induced a biphasic response that had a total duration of 2-15 min. An initial inward shift in holding current (I$\sb{\rm in}$), which lasted 5-120 sec, was followed by a slow outward shift in holding current (I$\sb{\rm out}$). At membrane potentials more negative than $-40$ mV, I$\sb{\rm in}$ was associated with a small and relatively voltage-independent increase in membrane conductance. I$\sb{\rm in}$ was not blocked by bath application of TTX or Co$\sp{2+}$. Although I$\sb{\rm in}$ was activated by injection of IP$\sb3$, it was not blocked by iontophoretic injection of ethyleneglycol-bis-(beta-aminoethyl ether), N, N$\sp\prime$-tetraacetic acid (EGTA) sufficient to block the Ca$\sp{2+}$-activated inward tail current (I$\sb{\rm B}$).^ Long-term (lasting at least 24 hours) effects of adenylate cyclase activation were examined in a well characterized class of mechanosensory neurons in Aplysia. The injected cells were analyzed 24 hours later by two-electrode voltage-clamp techniques. We found that K$\sp+$ currents of these cells were reduced 24 hours after injection of cAMP. The currents that were reduced by cAMP were very similar to those found to be reduced 24 hours after behavioral sensitization. These results suggest that cAMP is part of the intracellular signal that induces long-term sensitization in Aplysia. (Abstract shortened with permission of author.) ^

Analysis of endogenous retinoic acid receptor expression and function in murine melanoma cells

There have been numerous reports over the past several years on the ability of vitamin A analogs (retinoids) to modulate cell proliferation, malignant transformation, morphogenesis, and differentiation in a wide variety of cell types and organisms. Two families of nuclear retinoid-inducible, trans-acting, transcription-enhancing receptors that bear strong DNA sequence homology to thyroid and steroid hormone receptors have recently been discovered. The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) each have at least three types designated $\alpha,$ $\beta,$ and $\gamma,$ which are encoded by separate genes and expressed in a tissue and cell type-specific manner. We have been interested in the mechanism by which retinoids inhibit tumor cell proliferation and induce differentiation. As a model system we have employed several murine melanoma cell lines (S91-C2, K1735P, and B16-F1), which are sensitive to the growth-inhibitory and differentiation-inducing effects of RA, as well as a RA-resistant subclone of one of the cell lines (S91-C154), in order to study the role of the nuclear RARs in these effects. The initial phase of this project consisted of the characterization of the expression pattern of the three known RAR and RXR types in the murine melanoma cell lines in order to determine whether any differences exist which may elucidate a role for any of the receptors in RA-induced growth inhibition and differentiation. The novel finding was made that the RAR-$\beta$ gene is rapidly induced from undetectable levels by RA treatment at the mRNA and protein level, and that the induction of RAR-$\beta$ by other biologically active retinoids correlated with their ability to inhibit the growth of the highly RA-sensitive S91-C2 cell line. This suggests a role for RAR-$\beta$ in the growth inhibiting effect of retinoids. The second phase of this project involves the stable expression of RAR-$\beta$ in the S91-C2 cells and the RAR-$\beta$ receptor-null cell line, K1735P. These studies have indicated an inverse correlation between RAR-$\beta$ expression and proliferation rate. ^

Development of micro-electrospray mass spectrometry for ultra high sensitivity and application in the study of drugs of abuse on endogenous \lbrack Met\rbrack $\sp5$-enkephalin release

A micro-electrospray interface was developed specifically for the neurobiological applications described in this dissertation. Incorporation of a unique nano-flow liquid chromatography micro-electrospray "needle" into the micro-electrospray interface (micro-ES/MS) increased the sensitivity of the mass spectrometric assay by $\sim$1000 fold and thus permitted the first analysis of specific neuroactive compounds in brain extracellular fluid collected by in vivo microdialysis (Md).^ Initial in vivo data presented deals with the pharmacodynamics of a novel GABA$\sb{\rm B}$ antagonist and the availability of the compound in its parent (unmetabolized) form to the brain of the anesthetized rat. Next, the first structurally specific endogenous release of (Met) $\sp5$-enkephalin was demonstrated in unanesthetized freely-moving animals (release of $\sim$6.5 fmole of (Met) $\sp5$-enkephalin into the dialysate by direct neuronal depolarization). The Md/micro-ES/MS system was used to test the acute effects of drugs of abuse on the endogenous release of (Met) $\sp5$-enkephalin from the globus pallidus/ventral pallidum brain region in rats. Four drugs known to be abused by man (morphine, cocaine, methamphetamine and diazepam) were tested. Morphine and cocaine both elicited a two-fold or more increase in the release of (Met) $\sp5$-enkephalin over vehicle controls. Diazepam elicited a small decrease in (Met) $\sp5$-enkephalin levels and methamphetamine showed no significant effect on (Met) $\sp5$-enkephalin. These results imply that (Met) $\sp5$-enkephalin may be involved in the reward pathway of certain drugs of abuse. ^

Changes in serum lipids with change and fluctuation in body mass index and body fat distribution in Mexican Americans with type 2 diabetes

This dissertation was written in the format of three journal articles. Paper 1 examined the influence of change and fluctuation in body mass index (BMI) over an eleven-year period, on changes in serum lipid levels (total, HDL, and LDL cholesterol, triglyceride) in a population of Mexican Americans with type 2 diabetes. Linear regression models containing initial lipid value, BMI and age, BMI change (slope of BMI), and BMI fluctuation (root mean square error) were used to investigate associations of these variables with change in lipids over time. Increasing BMI over time was associated with gains in total and LDL cholesterol and triglyceride levels in women. Fluctuation of BMI was not associated with detrimental lipid profiles. These effects were independent of age and were not statistically significant in men. In Mexican-American women with type 2 diabetes, weight reduction is likely to result in more favorable levels of total and LDL cholesterol and triglyceride, without concern for possible detrimental effects of weight fluctuation. Weight reduction may not be as effective in men, but does not appear to be harmful either. ^ Paper 2 examined the associations of upper and total body fat with total cholesterol, HDL and LDL cholesterol, and triglyceride levels in the same population. Multilevel analysis was used to predict serum lipid levels from total body fat (BMI and triceps skinfold) and upper body fat (subscapular skinfold), while controlling for the effects of sex, age and self-correlations across time. Body fat was not strikingly associated with trends in serum lipid levels. However, upper body fat was strongly associated with triglyceride levels. This suggests that loss of upper body fat may be more important than weight loss in management of the hypertriglyceridemia commonly seen in type 2 diabetes. ^ Paper 3 was a review of the literature reporting associations between weight fluctuation and lipid levels. Few studies have reported associations between weight fluctuation and total, LDL, and HDL cholesterol and triglyceride levels. The body of evidence to date suggests that weight fluctuation does not strongly influence levels of total, LDL and HDL cholesterol and triglyceride. ^

The heparan sulfate-fibroblast growth factor signaling system in liver growth and function

The heparan sulfate (HS)-fibroblast growth factor (FGF) signaling system is a ubiquitous regulator that senses local environmental changes and mediates cell-to-cell communication. This system consists of three mutually interactive components. These are regulatory polypeptides (FGF), FGF receptor (FGFR) and heparan sulfate proteoglycans (FGFRHS). All four FGFR genes are expressed in the adult liver. Expression of the FGFR1–3 genes is generally associated with non-parenchymal cells while expression of the FGFR4 gene is associated with parenchymal hepatocytes. We showed that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (−/−) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7α-hydroxylase (Cyp7a), the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. These results indicated that FGFR4 was not directly involved in liver growth but exerted negative control on liver bile acid synthesis. This was confirmed in transgenic mice overexpressing the constitutively active human FGFR4 in livers. The transgenic mice exhibited decreased fecal bile acid excretion, bile acid pool size, and expression of Cyp7a. Introduction of this constitutively active human FGFR4 into FGFR4 (−/−) mice restored the inhibition of bile acid synthesis. Activation of the c-Jun N-terminal Kinase (JNK) pathway by FGFR4 correlated with the repressive effect on bile acid synthesis. ^ To determine whether FGFR4 played a broader role in liver-specific metabolic function, we examined the impact of both acute and chronic exposure to CCl 4 in FGFR4 (−/−) mice. Following acute CCl4 exposure, the FGFR4 (−/−) mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair, with no apparent effect on liver cell proliferation and restoration of cellularity. Chronic CCl4 exposure resulted in severe fibrosis in livers of FGFR4 (−/−) mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8 hr delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl 4-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. ^ Of the 23 FGF polypeptides, FGF1 and FGF2 are present at significant levels in the liver. To determine whether FGF1 and FGF2 played a role in CCl 4-induced liver injury and fibrosis, we examined the impact of both acute and chronic exposure to CCl4 in both wild-type and FGF1-FGF2 double-knockout mice. Following acute CCl4 exposure, FGF1(−/−)FGF2(−/−) mice exhibited accelerated liver injury, overall normal liver growth and repair, and decreased liver collagen α1(I) induction. Liver fibrosis resulting from chronic CCl4 exposure was markedly decreased in livers of FGF1(−/−)FGF2(−/−) mice compared to wild-type mice. This study suggests a role for FGF1 and FGF2 in hepatic fibrogenesis. ^ In summary, our three part study shows that specific components of the ubiquitous HS-FGF signaling family in the liver context interfaces with metabolite- and xenobiotic-controlled networks to regulate liver function, but has no apparent direct effect on liver cell growth. ^

Analyzing the function of myogenin in adult satellite cells through the construction of a myogenin conditional allele

Although mechanisms regulating the formation of embryonic skeletal muscle are well characterized, less is known about muscle formation in postnatal life. This disparity is unfortunate because the largest increases in skeletal muscle mass occur after birth. Adult muscle stem cells (satellite cells) appear to recapitulate the events that occur in embryonic myoblasts. In particular, the myogenic basic helix-loop-helix factors, which have crucial functions in embryonic muscle development, are assumed to have similar roles in postnatal muscle formation. Here, I test this assumption by determining the role of the myogenic regulator myogenin in postnatal life. Myogenin-null mice die at birth, necessitating the generation of floxed alleles of myogenin and the use of cre-recombinase lines to delete myogenin. Removing myogenin before embryonic muscle development resulted in myofiber deficiencies identical to those observed in myogenin-null mice. However, mice in which myogenin was deleted following embryonic muscle development had normal skeletal muscle, except for modest alterations in MRF4 and MyoD expression. Notably, myogenin-deleted mice were 30% smaller than controls, suggesting that myogenin's absence disrupted general body growth. These results suggest that skeletal muscle growth in postnatal life is controlled by mechanisms distinct from those occurring in embryonic muscle development. ^

Investigations of stress responses in Saccharomyces cerevisiae: Transcriptional control and heat shock protein function

The baker's yeast, Saccharomyces cerevisiae responds to the cytotoxic effects of elevated temperature (37-42°C) by activating transcription of ∼150 genes, termed heat shock genes, collectively required to compensate for the abundance of misfolded and aggregated proteins and various physiological modifications necessary for the cell to survive and grow at heat shock temperatures. An intriguing facet of the yeast heat shock response is the remarkable similarity it shares with the global remodeling that occurs in mammalian cells in response to numerous pathophysiological conditions including cancer and cardiovascular disease and thus provides an ideal model system. I have therefore investigated several novel features of stress signaling, transcriptional regulation, and physiology. Initial work focused on the characterization of SYM1, a novel heat shock gene in yeast which was demonstrated to be required for growth on the nonfermentable carbon source ethanol at elevated temperature, and to be the functional ortholog of the mammalian kidney disease gene, Mpv17. Additional work addressed the role of two proteins, the Akt-related kinase, Sch9, and Sse1, the yeast Hsp110 protein chaperone homolog, in signaling by protein kinase A, establishing Sse1 as a critical negative regulator of this pathway. Furthermore, I have demonstrated a role for Sse1 in biogenesis and stability of the stress-response transcription factor, Msn2; a finding that has been extended to include a select subset of additional high molecular weight proteins, suggesting a more global role for this chaperone in stabilizing the cellular proteome. The final emphasis of my doctoral work has included the finding that celastrol, a compound isolated from the plant family Celasfraceae, a component of traditional Chinese herbal medicine, can activate heat shock transcription factor (Hsf1) in yeast and mammalian cells through an oxidative stress mechanism. Celastrol treatment simultaneously activates both heat shock and oxidative stress response pathways, resulting in increased cytoprotection. ^

Joint effects of pulmonary function and aerobic power on survival in a cohort of healthy adults

Objective measurements of physical fitness and pulmonary function are related individually to long-term survival, both in healthy people and in those who are ill. These factors are furthermore known to be related to one another physiologically in people with pulmonary disease, because advanced pulmonary disease causes ventilatory limitation to exercise. Healthy people do not have ventilatory limitation to exercise, but rather have ventilatory reserve. The relationship between pulmonary function and exercise performance in healthy people is minimal. Exercise performance has been shown to modify the effect of pulmonary function on mortality in people with chronic obstructive pulmonary disease, but the relationship between these factors in healthy people has not been studied and is not known. The purpose of this study is to quantify the joint effects of pulmonary function and exercise performance as these bear on mortality in a cohort of healthy adults. This investigation is an historical cohort study over 20 years of follow-up of 29,624 adults who had complete preventive medicine, spirometry and treadmill stress examinations at the Cooper Clinic in Dallas, Texas.^ In 20 years of follow-up, there were 738 evaluable deaths. Forced expiratory volume in one second (FEV$\sb1$) percent of predicted, treadmill time in minutes percent of predicted, age, gender, body mass index, baseline smoking status, serum glucose and serum total cholesterol were all significant, independent predictors of mortality risk. There were no frank interactions, although age had an important increasing effect on the risk associated with smoking when other covariates were controlled for in a proportional-hazards model. There was no confounding effect of exercise performance on pulmonary function. In agreement with the pertinent literature on independent effects, each unit increase in FEV$\sb1$ percent predicted was associated with about eight tenths of a percent reduction in adjusted mortality rate. The concept of physiologic reserve is useful in interpretation of the findings. Since pulmonary function does not limit exercise tolerance in healthy adults, it is reasonable to expect that exercise tolerance would not modify the effect of pulmonary function on mortality. Epidemiologic techniques are useful for elucidating physiological correlates of mortality risk. ^

A STUDY ON THE FUNCTION OF 14-3-3SIGMA IN REGULATING CANCER ENERGY METABOLISM

Metabolic reprogramming has been shown to be a major cancer hallmark providing tumor cells with significant advantages for survival, proliferation, growth, metastasis and resistance against anti-cancer therapies. Glycolysis, glutaminolysis and mitochondrial biogenesis are among the most essential cancer metabolic alterations because these pathways provide cancer cells with not only energy but also crucial metabolites to support large-scale biosynthesis, rapid proliferation and tumorigenesis. In this study, we find that 14-3-3σ suppresses all these three metabolic processes by promoting the degradation of their main driver, c-Myc. In fact, 14-3-3s significantly enhances c-Myc poly-ubiquitination and subsequent degradation, reduces c-Myc transcriptional activity, and down-regulates c-Myc-induced metabolic target genes expression. Therefore, 14-3-3σ remarkably blocks glycolysis, decreases glutaminolysis and diminishes mitochondrial mass of cancer cells both in vitro and in vivo, thereby severely suppressing cancer bioenergetics and metabolism. As a result, a high level of 14-3-3σ in tumors is strongly associated with increased breast cancer patients’ overall and metastasis-free survival as well as better clinical outcomes. Thus, this study reveals a new role for 14-3-3s as a significant regulator of cancer bioenergetics and a promising target for the development of anti-cancer metabolism therapies.

Expression and function of $\beta$-1,4-galactosyltransferase during wild-type and T/{\it t\/} -mutant spermatogenesis

In the mouse, gamete recognition is mediated in part by the binding of sperm surface $\beta$1,4 galactosyltransferase (GalTase) to specific oligosaccharide residues on the zona pellucida ZP3. The expression of GalTase on the sperm surface is regulated by alleles within the distal segment of the T/t complex and results in a haploid-specific increase in GalTase expression on spermatids and sperm from t-bearing males, suggesting that differences in sperm GalTase activity may contribute to t-sperm transmission ratio distortion. In this study, the expression of GalTase RNA during wild-type and T/t-mutant spermatogenesis was characterized and the role of GalTase was analyzed in transmission ratio distortion. It was found that spermatogenic cells predominantly express the long form of the GalTase RNA, which encodes the GalTase protein that is preferentially targeted to the cell surface in somatic cells. In wild-type testes, GalTase RNA accumulates during the maturation of primary spermatocytes, reaches peak levels prior to meiosis, and decreases and meiosis. GalTase RNA accumulates to similar levels during the maturation of +/t and t/t primary spermatocytes, but unlike wild-type, the level of GalTase RNA in t-spermatocytes remains elevated during meiotic division. Consequently, spermatids in t-mutant testes inherit higher levels of GalTase RNA than do wild-type spermatids, which likely accounts for the haploid-specific increase in surface GalTase activity characteristic of spermatids from t-bearing mice.^ The functional significance of the increased GalTase activity during t-sperm transmission ratio distortion was determined by examining the distribution of GalTase RNA and surface GalTase protein in haploid spermatids from +/t males. Results show that +- and t-spermatids have similar levels of both GalTase RNA and protein, indicating that transmission ratio distortion in +/t mice is not likely due to haploid-specific differences in sperm surface GalTase activity.^ The presence of GalTase on the surface of an early spermatogenic cells before it is required on the mature sperm to perform its function during gamete binding suggests a separate function for GalTase in Sertoli-germ cell adhesion. Studies indicate that cell surface GalTase partly mediates the initial adhesion of pachytene spermatocytes, but not haploid spermatids, to Sertoli cells. ^

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. ^