Faculty Bibliography

Cholesterol is a major component of the plasma membrane in mammalian cells, where it acts as a modulator of bulk physical state and integrity. In addition to its structural role, cholesterol is essential for proliferation and other cell processes. The present study was undertaken to explore the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression. Comparisons were made between cholesterol and other sterol analogs that differ from cholesterol in three specific elements: the presence of a Delta5 double bond in ring B, the hydroxyl group at C-3, and the presence of an aliphatic side chain. The human leukemia cells HL-60 and MOLT-4 were cultured in cholesterol-free medium and treated with different sterols in the presence or absence of SKF 104976, a competitive inhibitor of lanosterol 14alpha-demethylase that allows the synthesis of isoprenoid derivatives but not cholesterol. Our results show that the beta-hydroxyl group at C-3 and the unsaturated bond at Delta5 are necessary for cell proliferation and cell cycle progression. The sterol analog 5alpha-cholestan-3beta-ol (dihydrocholesterol), which is saturated at Delta5 and has an A/B ring junction in the trans configuration, was also able to support cell growth. However, 5beta-cholestan-3beta-ol and 5beta-cholestan-3alpha-ol, both of which have an A/B ring junction in the cis configuration, were totally ineffective in supporting cell growth. Indeed, they produced an inhibition of cell proliferation and arrested the cell cycle specifically in the G2/M phase. These effects of 5beta-cholestanols were abrogated by cholesterol in a concentration-dependent manner. Moreover, 5beta-cholestanols potently inhibited cholesterol biosynthesis and transcription driven by the sterol response element. In addition to providing a description of the structural features of sterols associated with their supporting action on cell proliferation in mammalian cells, the present results demonstrate that selected cholesterol analogs may act as cytostatic agents, interrupting cell cycle progression specifically in the G2/M phase

Transforming growth factor-beta is released from most cells as an inactive complex consisting of transforming growth factor-beta, the transforming growth factor-beta propeptide and the latent transforming growth factor-beta-binding protein. We studied the role of latent transforming growth factor-beta-binding protein in modulating transforming growth factor-beta availability by generating transgenic mice that express a truncated form of latent transforming growth factor-beta-binding protein-1 that binds to transforming growth factor-beta but is missing the known N- and C-terminal matrix-binding sequences. As transforming growth factor-beta is an inhibitor of keratinocyte proliferation and is involved in the control of hair cycling, we over-expressed the mutated form of latent transforming growth factor-beta-binding protein under the control of the keratin 14-promoter. Transgenic animals displayed a hair phenotype due to a reduction in keratinocyte proliferation, an abbreviated growth phase and an early initiation of the involution (catagen) phase of the hair cycle. This phenotype appears to result from excess active transforming growth factor-beta, as enhanced numbers of pSmad2/3-positive nuclei are observed in transgenic animal skin. These data suggest that the truncated form of latent transforming growth factor-beta-binding protein-1 competes with wild-type latent transforming growth factor-beta-binding protein for binding to latent transforming growth factor-beta, resulting in latent transforming growth factor-beta complexes that fail to be targeted correctly in the extracellular matrix. The mis-localization of the transforming growth factor-beta results in inappropriate activation and premature initiation of catagen, thereby illustrating the significance of latent transforming growth factor-beta-binding protein interaction with transforming growth factor-beta in the targeting and activation of latent transforming growth factor-beta in addition to previously reported effects on small latent complex secretion

Transgenic mouse models have been essential for understanding the pathogenesis of Alzheimer's disease (AD) including those that model the deposition process of beta-amyloid (Abeta). Several laboratories have focused on research related to the non-invasive detection of early changes in brains of transgenic mouse models of Alzheimer's pathology. Most of this work has been performed using regional image analysis of individual mouse brains and pooling the results for statistical assessment. Here we report the implementation of a non-linear image registration algorithm to register anatomical and transverse relaxation time (T2) maps estimated from MR images of transgenic mice. The algorithm successfully registered mouse brain magnetic resonance imaging (MRI) volumes and T2 maps, allowing reliable estimates of T2 values for different regions of interest from the resultant combined images. This approach significantly reduced the data processing and analysis time, and improved the ability to statistically discriminate between groups. Additionally, 3D visualization of intra-regional distributions of T2 of the resultant registered images provided the ability to detect small changes between groups that otherwise would not be possible to detect

BACKGROUND: Trigeminal sensory neurons detect thermal and mechanical stimuli in the skin through their elaborately arborized peripheral axons. We investigated the developmental mechanisms that determine the size and shape of individual trigeminal arbors in zebrafish and analyzed how these interactions affect the functional organization of the peripheral sensory system. RESULTS: Time-lapse imaging indicated that direct repulsion between growing axons restricts arbor territories. Removal of one trigeminal ganglion allowed axons of the contralateral ganglion to cross the midline, and removal of both resulted in the expansion of spinal cord sensory neuron arbors. Generation of embryos with single, isolated sensory neurons resulted in axon arbors that possessed a vast capacity for growth and expanded to encompass the entire head. Embryos in which arbors were allowed to aberrantly cross the midline were unable to respond in a spatially appropriate way to mechanical stimuli. CONCLUSIONS: Direct repulsive interactions between developing trigeminal and spinal cord sensory axon arbors determine sensory neuron organization and control the shapes and sizes of individual arbors. This spatial organization is crucial for sensing the location of objects in the environment. Thus, a combination of undirected growth and mutual repulsion results in the formation of a functionally organized system of peripheral sensory arbors

BACKGROUND: Medical and pre-professional health students ask questions about human health that can be answered in two ways, by giving proximate and evolutionary explanations. Proximate explanations, most common in textbooks and classes, describe the immediate scientifically known biological mechanisms of anatomical characteristics or physiological processes. These explanations are necessary but insufficient. They can be complemented with evolutionary explanations that describe the evolutionary processes and principles that have resulted in human biology we study today. The main goal of the science of Darwinian Medicine is to investigate human disease, disorders, and medical complications from an evolutionary perspective. DISCUSSION: This paper contrasts the differences between these two types of explanations by describing principles of natural selection that underlie medical questions. Thus, why is human birth complicated? Why does sickle cell anemia exist? Why do we show symptoms like fever, diarrhea, and coughing when we have infection? Why do we suffer from ubiquitous age-related diseases like arteriosclerosis, Alzheimer's and others? Why are chronic diseases like type II diabetes and obesity so prevalent in modern society? Why hasn't natural selection eliminated the genes that cause common genetic diseases like hemochromatosis, cystic fibrosis, Tay sachs, PKU and others? SUMMARY: In giving students evolutionary explanations professors should underscore principles of natural selection, since these can be generalized for the analysis of many medical questions. From a research perspective, natural selection seems central to leading hypotheses of obesity and type II diabetes and might very well explain the occurrence of certain common genetic diseases like cystic fibrosis, hemochromatosis, Tay sachs, Fragile X syndrome, G6PD and others because of their compensating advantages. Furthermore, armed with evolutionary explanations, health care professionals can bring practical benefits to patients by treating their symptoms of infection more specifically and judiciously. They might also help curtail the evolutionary arms race between pathogens and antibiotic defenses

A dramatic overexpression of IGFBP-1 is responsible for growth inhibition, in response to a low-protein diet feeding. It has been demonstrated that a fall in the amino acid concentration was directly responsible for IGFBP-1 induction. In this report, we sought to determine the mechanism by which amino acid limitation upregulates IGFBP-1 expression. Our results show that both transcriptional activation and mRNA stabilization are involved. We also demonstrate that (i) the mGCN2/ATF4 pathway is not involved in this regulation and (ii) the 3'UTR of IGFBP-1 mRNA is responsible for its destabilization and regulates its stability in response to amino acid starvation.

Genetic deficiency of the plasma phospholipid transfer protein (PLTP) in mice unexpectedly causes a substantial impairment in liver secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins. To explore the mechanism, we examined the three known pathways for hepatic apoB secretory control, namely endoplasmic reticulum (ER)/proteasome-associated degradation (ERAD), post-ER pre-secretory proteolysis (PERPP), and receptor-mediated degradation, also known as re-uptake. First, we found that ERAD and cell surface re-uptake were not active in PLTP-null hepatocytes. Moreover, ER-to-Golgi blockade by brefeldin A, which enhances ERAD, equalized total apoB recovery from PLTP-null and wild-type cells, indicating that the relevant process occurs post-ER. Second, because PERPP can be stimulated by intracellular reactive oxygen species (ROS), we examined hepatic redox status. Although we found previously that PLTP-null mice exhibit elevated plasma concentrations of vitamin E, a lipid anti-oxidant, we now discovered that their livers contain significantly less vitamin E and significantly more lipid peroxides than do livers of wild-type mice. Third, to establish a causal connection, the addition of vitamin E or treatment with an inhibitor of intracellular iron-dependent peroxidation, desferrioxamine, abolished the elevation in cellular ROS as well as the defect in apoB secretion from PLTP-null hepatocytes. Overall, we conclude that PLTP deficiency decreases liver vitamin E content, increases hepatic oxidant tone, and substantially enhances ROS-dependent destruction of newly synthesized apoB via a post-ER process. These findings are likely to be broadly relevant to hepatic apoB secretory control in vivo.

Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis