The impact of functional impairment on the survival of patients with Alzheimer's disease

Alzheimer's disease (AD), the most common form of dementia, is the fifth leading cause of death among U.S. adults aged 65 or older. Most AD patients have shorter life expectancy compared with older people without dementia. This disease has become an enormous challenge in the aging society and is also a global problem. Not only do families of patients with Alzheimer's disease need to pay attention to this problem, but also the healthcare system and society as a whole have to confront. In dementia, functional impairment is associated with basic activities of daily living (ADL) and instrumental activities of daily living (IADL). For patients with Alzheimer's disease, problems typically appear in performing IADL and progress to the inability of managing less complex ADL functions of personal care. Thus, assessment of ADLs can be used for early accurate diagnosis of Alzheimer's disease. It should be useful for patients, caregivers, clinicians, and policy planners to estimate the survival of patients with Alzheimer's disease. However, it is unclear that when making predictions of patient outcome according to their histories, time-dependent covariates will provide us with important information on how changes in a patient's status can effect the survival. In this study, we examined the effect of impaired basic ADL as measured by the Physical Self-Maintenance Scale (PSMS) and utilized a multistate survival analysis approach to estimate the probability of death in the first few years of initial visit for AD patients taking into consideration the possibility of impaired basic ADL. The dataset used in this study was obtained from the Baylor Alzheimer's Disease and Memory Disorders Center (ADMDC). No impaired basic ADL and older age at onset of impaired basic ADL were associated with longer survival. These findings suggest that the occurrence of impaired basic ADL and age at impaired basic ADL could be predictors of survival among patients with Alzheimer's disease. ^

Spectroscopic and functional investigations of the AMPA subtype of ionotropic glutamate receptors

Ion channels play a crucial role in the functioning of different systems of the body because of their ability to bridge the cell membrane and allow ions to pass in and out of the cell. Ionotropic glutamate receptors are one class of these important proteins and have been shown to be critical in propagating synaptic transmission in the central nervous system and in other diverse functions throughout the body. Because of their wide-ranging effects, this family of receptors is an important target for structure-function investigations to understand their mechanism of action. ^ α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one subtype of glutamate receptors and have been shown to be the primary receptors involved in rapid excitatory signaling in the central nervous system. Agonist binding to the extracellular ligand binding domain of these receptors causes various conformational changes that culminate in formation of the ion channel. Previous structural investigations have provided important information about their mechanism of action, including uncovering a relationship between the degree of cleft closure in the binding domain and activation of the receptor. However, what question remains unanswered is how specific interactions between the agonist and the protein interplay with cleft closure to mediate receptor activation. ^ To investigate this question, I applied a multiscale approach to investigate the effects of agonist binding on various levels. Vibrational spectroscopy was utilized to investigate molecular-level interactions in the binding pocket, and fluorescence resonance energy transfer (FRET) was employed to measure cleft closure in the isolated ligand binding domain. The results of these studies in the isolated binding domain were then correlated to activation of the full receptor. These investigations showed a relationship between the strength of the interaction at the α-amine group of the agonist and extent of receptor activation, where a stronger interaction correlated to a larger activation, which was upheld even when the extent of cleft closure did not correlate to activation. These results show that this interaction at the α-amine group is critical in mediating the allosteric mechanism of activation and provide a bit more insight into how agonist binding is coupled to channel gating in AMPA receptors. ^

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform.

The structural and functional relationship of the p53 tumor suppressor protein

The tumor-suppressing function of p53 can be affected in a variety of manners. Here, we describe a novel mechanism of transformation by mutant p53. Previously, it had been believed that mutant p53 molecules transform cells by oligomerizing with wild-type p53 and inactivating it. However, we demonstrated that there exists an additional mechanism of inactivation of p53 available to p53 mutants. It involves sequestration of cofactors necessary to p53, and subsequent interruption of its transactivation and tumor suppression functions. The p53 amino or carboxyl termini, known to interact with a large number of cellular factors, can affect wild-type p53 in this manner. Although they are unable to oligomerize with wild-type p53, they transform cells containing p53, and inhibit its transactivation ability. In addition, they interrupt growth suppression by p53, but not RB, confirming that they specifically affect p53 function, rather than having a general growth-stimulatory phenomenon. Also, we have cloned a p53 tumor mutation which results in expression of the amino terminus of p53. This provides a means to study the factor-sequestration transforming mechanism in vivo. Additionally, we found that the published sequence of the mdm2 gene is in error. mdm2 is a gene intimately involved with p53, blocking its ability to transform cells. Finally, previous data had established the influence of cell-cycle status on p53 function. In growth-arrested cells, wild-type p53 expressed by a transgene cannot activate transcription, but if these cells are forced to cycle by addition of cyclin E, p53 once again becomes functional. In this study, we extend these findings by examining only those cells successfully transfected, using fluorescence-activated cell sorting. Our results support the previous data, that cyclin E pushes growth-arrested cells back into the cell cycle. In summary, we have demonstrated the potential importance of cofactor association and protein modification to the abilities of p53 to cause transcription activation and repression, inhibition of DNA replication and induction of DNA repair, and initiation of cell-cycle arrest and apoptosis. Further elucidation of these processes and their roles in tumor suppression will prove fascinating indeed. ^

Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

The functional role of the tumor suppressor MMAC /PTEN in glioblastoma multiforme and prostate cancer

Investigations into the molecular basis of glioblastoma multiforme led to the identification of a putative tumor suppressor gene, MMAC/ PTEN. Initial studies implicated MMAC/PTEN in many different tumor types, and identified a protein phosphatase motif in its sequence. This project aimed to identify the biological and biochemical functions of MMAC/PTEN by transiently expressing the gene in cancer cells that lack a functional gene product. ^ Expression of MMAC/PTEN mildly suppressed the growth of U251 human glioma cells and abrogated the growth advantage mediated by overexpression of the epidermal growth factor receptor (EGFR). Immunoblotting demonstrated that MMAC/PTEN expression did not affect the phosphorylation of the EGFR itself, or the intermediates of several downstream signaling pathways. However, MMAC/PTEN expression significantly reduced the phosphorylation and catalytic activity of the proto-oncogene Akt/PKB. While Akt/PKB regulates the survival of many cell types, expression of MMAC/PTEN did not induce apoptosis in adherent U251 cells. Instead, MMAC/PTEN expression sensitized the cells to apoptosis when maintained in suspension (anoikis). As the survival of suspended cells is one of the hallmarks leading to metastasis, MMAC/PTEN expression was examined in a system in which metastasis is more clinically relevant, prostate cancer. ^ Expression of MMAC/PTEN in both LNCaP and PC3-P human prostate cancer cells specifically inhibited Akt/PKB phosphorylation. MMAC/PTEN expression in LNCaP cells resulted in a profound inhibition of growth that was significantly greater than that achieved with expression of p53. Expression of MMAC/PTEN in PC3-P cells resulted in greater growth inhibition than was observed in U251 glioma cells, but less than was observed in LNCaP cells, or upon p53 expression. To determine if MMAC/PTEN could function as a tumor suppressor in vivo, the effects of MMAC/PTEN expression on PC3-P cells implanted orthotopically in nude mice were examined. The ex-vivo expression of MMAC/PTEN did not decrease tumor incidence, but it did significantly decrease tumor size and metastasis. In-vivo expression of MMAC/PTEN in pre-established PC3-P tumors did not significantly inhibit tumor incidence or size, but did inhibit metastasis formation. ^ These studies demonstrate that MMAC/PTEN is a novel and important tumor suppressor gene, which functions to downregulate an important cell survival signaling pathway. ^

Functional analysis of the Galpha protein, GNA -3, in the development of Neurospora crassa using biochemical and genetic studies

Extracellular signals regulate fungal development and, to sense and respond to these cues, fungi evolved signal transduction pathways similar to those in mammalian systems. In fungi, heterotrimeric G proteins, composed of α, β, and γ subunits, transduce many signals, such as pheromones and nutrients, intracellularly to alter adenylyl cyclase and MAPK cascades activity. ^ Previously, the Gα proteins GNA-1 and GNA-2 were characterized in regulating development in the fungus Neurospora crassa. R. A. Baasiri isolated a third Gα, gna-3, and P. S. Rowley generated Δgna-3 mutants. GNA-3 belongs to a fungal Gα family that regulates cAMP metabolism and virulence. The Δ gna-3 sexual cycle is defective in homozygous crosses, producing inviable spores. Δgna-3 mutants have reduced aerial hyphae formation and derepressed asexual sporulation (conidiation), causing accumulation of asexual spores (conidia). These defects are similar to an adenylyl cyclase mutant, cr-1; cAMP supplementation suppressed Δ gna-3 and cr-1. Inappropriate conidiation and expression of a conidiation gene, con-10, were higher in Δ gna-3 than cr-1 submerged cultures; peptone suppressed conidiation. Adenylyl cyclase activity and expression demonstrated that GNA-3 regulates enzyme levels. ^ A Δgna-1 cr-1 was analyzed with F. D. Ivey to differentiate GNA-1 roles in cAMP-dependent and -independent pathways. Δ gna-1 cr-1 defects were worse than cr-1 and refractory to cAMP, suggesting that GNA-1 is necessary for sensing extracellular CAMP. Submerged culture conidiation was highest in Δgna-1 cr-1, and only high cell density Δgna-1 cultures conidiated, which correlated with con-10 levels. Transcription of a putative heat shock cognate protein was highest in Δgna-1 cr-1. ^ Functional relationships between the three Gαs was analyzed by constructing Δgna-1 Δgna-2 Δ gna-3, Δgna-1 Δgna-3, and Δgna-2 Δgna-3 strains. Δ gna-2 Δgna-3 strains exhibited intensified Δ gna-3 phenotypes; Δgna-1 Δgna-2 Δgna-3 and Δgna-1 Δ gna-3 strains were identical to Δgna-1 cr-1 on plates and were non-responsive to cAMP. The highest levels of conidiation and con-10 were detected in submerged cultures of Δ gna-1 Δgna-2 Δgna-3 and Δgna-1 Δgna-3 mutants, which was partially suppressed by peptone supplementation. Stimulation of adenylyl cyclase is completely deficient in Δgna-1 Δ gna-2 Δgna-3 and Δgna-1 Δ gna-3 strains. Δgna-3 and Δ gna-1 Δgna-3 aerial hyphae and conidiation defects were suppressed by mutation of a PKA regulatory subunit. ^

A novel motion tracking system for evaluation of functional rehabilitation of the upper limbs

Upper limb function impairment is one of the most common sequelae of central nervous system injury, especially in stroke patients and when spinal cord injury produces tetraplegia. Conventional assessment methods cannot provide objective evaluation of patient performance and the tiveness of therapies. The most common assessment tools are based on rating scales, which are inefficient when measuring small changes and can yield subjective bias. In this study, we designed an inertial sensor-based monitoring system composed of five sensors to measure and analyze the complex movements of the upper limbs, which are common in activities of daily living. We developed a kinematic model with nine degrees of freedom to analyze upper limb and head movements in three dimensions. This system was then validated using a commercial optoelectronic system. These findings suggest that an inertial sensor-based motion tracking system can be used in patients who have upper limb impairment through data integration with a virtual reality-based neuroretation system.

Optical imaging of functional domains in the cortex of the awake and behaving monkey

As demonstrated by anatomical and physiological studies, the cerebral cortex consists of groups of cortical modules, each comprising populations of neurons with similar functional properties. This functional modularity exists in both sensory and association neocortices. However, the role of such cortical modules in perceptual and cognitive behavior is unknown. To aid in the examination of this issue we have applied the high spatial resolution optical imaging methodology to the study of awake, behaving animals. In this paper, we report the optical imaging of orientation domains and blob structures, approximately 100–200 μm in size, in visual cortex of the awake and behaving monkey. By overcoming the spatial limitations of other existing imaging methods, optical imaging will permit the study of a wide variety of cortical functions at the columnar level, including motor and cognitive functions traditionally studied with positron-emission tomography or functional MRI techniques.

Functional characterization of the C—C chemokine-like molecules encoded by molluscum contagiosum virus types 1 and 2

Many viruses have evolved mechanisms for evading the host immune system by synthesizing proteins that interfere with the normal immune response. The poxviruses are among the most accomplished at deceiving their hosts’ immune systems. The nucleotide sequence of the genome of the human cutaneous poxvirus, molluscum contagiosum virus (MCV) type 1, was recently reported to contain a region that resembles a human chemokine. We have cloned and expressed the chemokine-like genes from MCV type 1 and the closely related MCV type 2 to determine a potential role for these proteins in the viral life cycle. In monocyte chemotaxis assays, the viral proteins have no chemotactic activity but both viral proteins block the chemotactic response to the human chemokine, macrophage inflammatory protein (MIP)-1α. Like MIP-1α, both viral proteins also inhibit the growth of human hematopoietic progenitor cells, but the viral proteins are more potent in this activity than the human chemokine. These viral chemokines antagonize the chemotactic activity of human chemokines and have an inhibitory effect on human hematopoietic progenitor cells. We hypothesize that the inhibition of chemotaxis is an immune evasion function of these proteins during molluscum contagiosum virus infection. The significance of hematopoietic progenitor cell inhibition in viral pathogenesis is uncertain.

Functional cleavage of the common cytokine receptor γ chain (γc) by calpain

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor γ chain (γc) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleave in vitro-translated wild-type γc, but not γc containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of γc in a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of γc. Moreover, in single positive CD4+ thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to γc blocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of γc represents a mechanism by which γc-dependent signaling can be controlled.

Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor β2 (TGF-β). We have determined its crystal structure at a resolution of 2.5 Å, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-β. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional “hot spots” composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-β. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.

An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of ≈50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome ≈30 million years ago.

Functional breakdown of the lipid bilayer of the myelin membrane in central and peripheral nervous system by disrupted galactocerebroside synthesis

The lipid bilayer of the myelin membrane of the central nervous system (CNS) and the peripheral nervous system (PNS) contains the oligodendrocyte- and Schwann cell-specific glycosphingolipids galactocerebrosides (GalC) and GalC-derived sulfatides (sGalC). We have generated a UDP-galactose ceramide galactosyltransferase (CGT) null mutant mouse (cgt−/−) with CNS and PNS myelin completely depleted of GalC and derived sGalC. Oligodendrocytes and Schwann cells are unable to restore the structure and function of these galactosphingolipids to maintain the insulator function of the membrane bilayer. The velocity of nerve conduction of homozygous cgt−/− mice is reduced to that of unmyelinated axons. This indicates a severely altered ion permeability of the lipid bilayer. GalC and sGalC are essential for the unperturbed lipid bilayer of the myelin membrane of CNS and PNS. The severe dysmyelinosis leads to death of the cgt−/− mouse at the end of the myelination period.

Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic α-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly 15N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane α-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu2–51 (residues 2–51), which contains the transmembrane α-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu28–81 (residues 28–81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2–81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ± 3 pS and 12 ± 3 pS in 0.5 M KCl and 29 ± 3 pS and 12 ± 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu2–51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu28–81, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.