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1.  Induction of Fetal Globin in β-Thalassemia: Cellular Obstacles and Molecular Progress 
Accelerated apoptosis of erythroid progenitors in β-thalassemia is a significant barrier to definitive therapy because the beneficial effects of fetal globin–inducing agents on globin chain balance may not be inducible in cells in which programmed cell death is established early. Accordingly, our objectives have been to identify methods to decrease cellular apoptosis and to identify orally tolerable fetal globin gene inducers. A pilot clinical trial was conducted to determine whether combined use of a fetal globin gene inducer (butyrate) and rhu-erythropoietin (EPO), the hematopoietic growth factor that prolongs erythroid cell survival and stimulates erythroid proliferation, would produce additive hematologic responses in any thalassemia subjects. Butyrate and EPO were administered in 10 patients. Novel fetal globin gene inducers that also stimulate erythroid proliferation were evaluated for pharmacokinetic profiles. Patients with β+-thalassemia had relatively low levels of endogenous EPO (<145 mU/mL) and had additive responses to administered EPO and butyrate. Patients with at least one β0 -globin mutation had higher baseline HbF levels (>60%) and EPO levels (>160 mU/mL), and three-fourths of these subjects responded to the fetal globin gene inducer alone. A few select fetal globin–inducing short-chain fatty acid derivatives that stimulated cell proliferation also had favorable pharmacokinetics. These studies identify a significant subset of thalassemia patients who appear to require exogenous EPO to respond optimally to any HbF inducer, as well as new therapeutic candidates that act on both cellular and molecular pathologies of β-thalassemia. Both approaches now offer excellent potential for tolerable, definitive treatment of β-thalassemia.
doi:10.1196/annals.1345.033
PMCID: PMC4261694  PMID: 16339673
thalassemia; short-chain fatty acid; erythropoietin; fetal hemoglobin; apoptosis; molecular signaling
2.  Hemolysis-Associated Pulmonary Hypertension in Thalassemia 
Accumulating evidence supports the existence of a condition involving hemolysis-associated pulmonary hypertension. We reported in sickle cell disease hemolysis induced release of cell-free hemoglobin, and red blood cell arginase resulting in impaired nitric oxide bioavailability, endothelial dysfunction, and pulmonary hypertension. Since thalassemia is also a condition of chronic hemolysis, these patients are at risk. Our data demonstrates that hemolysis-induced dysregulation of arginine metabolism and pulmonary hypertension also occurs in thalassemia. Erythrocyte release of arginase during hemolysis contributes to the development of pulmonary hypertension. Therapies that maximize arginine and nitric oxide bioavailability may benefit patients with thalassemia.
doi:10.1196/annals.1345.058
PMCID: PMC3217300  PMID: 16339702
3.  Sampling Effort/Frequency Necessary to Infer Individual Acute Stress Responses from Fecal Analysis in Greylag Geese (Anser anser) 
Measuring hormone metabolites from excreta is a powerful method to study hormone–behavior relationships. Currently, fecal corticosterone metabolite concentrations are used to estimate individual short-term stress responses. From the free-roaming, semitame flock of greylag geese (Anser anser), as many fecal samples as possible were collected over 3 h following a challenge (social density stress) or in a control situation. This time span corresponds to the gut passage time of geese. It was asked how many samples were necessary to determine differences in excreted corticosterone immunoreactive metabolites (CORTs) between control and social density stress and which parameters (means, maxima, range) reliably showed this difference. A large variation of CORT was found between consecutive samples. Still, means, maxima, and ranges of the samples in a fecal series consistently showed the response to a stressor both within and between individuals. Three samples sufficed if the maximum value of CORT was used, whereas four or more samples were necessary to work with the mean. It was concluded that by increasing the number of fecal samples collected, the course of CORT could be measured more precisely and an individual’s acute stress response inferred more reliably.
doi:10.1196/annals.1343.012
PMCID: PMC3182528  PMID: 16055849
corticosterone–immunoreactive metabolites; sampling effort; noninvasive sampling; individual variation; short-term stress response; greylag goose; Anser anser
4.  Neurodistribution of Androgen Receptor Immunoreactivity in the Male Frog, Rana esculenta 
Sexual behavior in vertebrates depends on the cyclic release of steroids and their binding to the brain receptors. Previously, we demonstrated the presence of specific binding of 3H-testosterone and staining with PG-21 in the brain of the adult male frog, Rana esculenta. Here, we report our further receptor characterization using an anti–androgen receptor antiserum, PG-21, and the androgen site of action in frog brain. Nuclei, which contained cells labeled for the androgen receptor (AR), were mainly identified in the olfactory bulbs, preoptic-septal region, infundibulum, amygdala, thalamus, tectum, torus semicircularis, and medulla. The neuroanatomical AR staining appears similar to that in other lower vertebrates.
doi:10.1196/annals.1327.054
PMCID: PMC2967251  PMID: 15891054
androgen receptor; amphibian; brain; PG-21
5.  Role of Glial Cell Line–Derived Neurotrophic Factor in Germ-Line Stem Cell Fate 
The overall goal of this study is to unravel the role(s) played by glial cell line-derived neurotrophic factor (GDNF) in the fate of spermatogonial stem cells. There is great interest in the biology of spermatogonial stem cells, or Asingle spermatogonia, because of their importance in the treatment of infertility, the development of contraceptives, and the understanding of the etiology of testicular cancer, particularly seminoma. In the mouse, spermatogonial stem cells express GFRα-1, the receptor for GDNF, and respond to this growth factor in vivo and in vitro. GDNF is produced by the adjacent Sertoli cells, which are part of the germ-line stem cell niche in vertebrates. We specifically isolated GFRα-1–positive spermatogonia using an immunomagnetic bead technique. We then stimulated the cells with 100 ng/mL of rGDNF for 10 hours; unstimulated cells served as negative controls. Microarray analysis, immunocytochemistry, and Western blotting revealed that Numb, a regulator of the Notch pathway, is upregulated by GDNF in spermatogonial stem cells. There are indications that in rats, mice, and humans, the Notch pathway promotes spermatogonial differentiation. We observed that an increase in Numb expression is concomitant with Notch degradation in these cells. Thus, through Numb, GDNF might inhibit differentiation and allows the maintenance of the stem cell pool in the mouse seminiferous epithelium.
doi:10.1196/annals.1336.010
PMCID: PMC2904487  PMID: 16467260
GDNF; Notch; Numb; spermatogonia; testis stem cells
6.  Physiology and Pathophysiology of Iron Cardiomyopathy in Thalassemia 
Iron cardiomyopathy remains the leading cause of death in patients with thalassemia major. Magnetic resonance imaging (MRI) is ideally suited for monitoring thalassemia patients because it can detect cardiac and liver iron burdens as well as accurately measure left ventricular dimensions and function. However, patients with thalassemia have unique physiology that alters their normative data. In this article, we review the physiology and pathophysiology of thalassemic heart disease as well as the use of MRI to monitor it. Despite regular transfusions, thalassemia major patients have larger ventricular volumes, higher cardiac outputs, and lower total vascular resistances than published data for healthy control subjects; these hemodynamic findings are consistent with chronic anemia. Cardiac iron overload increases the relative risk of further dilation, arrhythmias, and decreased systolic function. However, many patients are asymptomatic despite heavy cardiac burdens. We explore possible mechanisms behind cardiac iron-function relationships and relate these mechanisms to clinical observations.
doi:10.1196/annals.1345.047
PMCID: PMC2892916  PMID: 16339687
iron; heart; MRI; ejection fraction; cardiac function; T2*
7.  Multiscale Modeling of Cardiac Cellular Energetics 
Multiscale modeling is essential to integrating knowledge of human physiology starting from genomics, molecular biology, and the environment through the levels of cells, tissues, and organs all the way to integrated systems behavior. The lowest levels concern biophysical and biochemical events. The higher levels of organization in tissues, organs, and organism are complex, representing the dynamically varying behavior of billions of cells interacting together. Models integrating cellular events into tissue and organ behavior are forced to resort to simplifications to minimize computational complexity, thus reducing the model’s ability to respond correctly to dynamic changes in external conditions. Adjustments at protein and gene regulatory levels shortchange the simplified higher-level representations. Our cell primitive is composed of a set of subcellular modules, each defining an intracellular function (action potential, tricarboxylic acid cycle, oxidative phosphorylation, glycolysis, calcium cycling, contraction, etc.), composing what we call the “eternal cell,” which assumes that there is neither proteolysis nor protein synthesis. Within the modules are elements describing each particular component (i.e., enzymatic reactions of assorted types, transporters, ionic channels, binding sites, etc.). Cell subregions are stirred tanks, linked by diffusional or transporter-mediated exchange. The modeling uses ordinary differential equations rather than stochastic or partial differential equations. This basic model is regarded as a primitive upon which to build models encompassing gene regulation, signaling, and long-term adaptations in structure and function. During simulation, simpler forms of the model are used, when possible, to reduce computation. However, when this results in error, the more complex and detailed modules and elements need to be employed to improve model realism. The processes of error recognition and of mapping between different levels of model form complexity are challenging but are essential for successful modeling of large-scale systems in reasonable time. Currently there is to this end no established methodology from computational sciences.
doi:10.1196/annals.1341.035
PMCID: PMC2864600  PMID: 16093514
cardiac metabolic systems modeling; constraint-based analysis; energetics; multicellular tissues; oxidative phosphorylation
8.  Relaxin Stimulates Multiple Signaling Pathways 
Relaxin has been shown previously to stimulate cyclic AMP production and the activation of MAPK. We reported that phosphoinositide-3 kinase (PI3K) activity is required for biphasic stimulation of cAMP by relaxin and that relaxin treatment increased PI3K activity in THP-1 cells. A downstream target of PI3K is protein kinase C zeta (PKCζ). Relaxin stimulated translocation of PKCζ to the plasma membrane in THP-1, MCF-7, pregnant human myometrial (PHM1-31), and mouse mesangial (MMC) cells. PKCζ translocation is PI3K dependent and independent of cAMP production. Pharmacological and antisense approaches, utilized to inhibit or knock down PKCζ, resulted in a 40% inhibition of relaxin-stimulated cAMP production. The stimulation of PKCζ by relaxin therefore is downstream of PI3K leading to increased cAMP production. To determine the role of PI3K/PKCζ stimulation by relaxin on downstream-mediated events, we examined the increase in vascular endothelial growth factor (VEGF) gene expression by relaxin. Treatment of THP-1 or MMC cells with the PI3K inhibitor, LY294002, abolished the relaxin-mediated stimulation of VEGF transcript levels. In summary, relaxin has pleiotropic signaling effects in THP-1 cells activating ERK1/2, cAMP, PI3K, and PKCζ. We have described a novel bifurcated pathway by which relaxin stimulates Gs alpha and PI3K/PKCζ leading to increased cAMP production and increased VEGF gene expression. Some, but not all, of these pathways are detected in other cell lines which may cause the unique diversity of downstream responses from this interesting hormone.
doi:10.1196/annals.1282.040
PMCID: PMC2846546  PMID: 15956717
adenylyl cyclase; PI3K; PKC zeta; relaxin; cyclic AMP
9.  Relaxin Stimulates cAMP Production in MCF-7 Cells upon Overexpression of Type V Adenylyl Cyclase 
Relaxin stimulates cAMP production and activation of ERK and PI3K in THP-1 cells. Relaxin also stimulates protein kinase C zeta (PKCζ) translocation to the plasma membrane in a PI3K-dependent manner in THP-1 and MCF-7 cells. However, relaxin did not increase cAMP production in MCF-7 cells. We overexpressed different adenylyl cyclase (AC) isoforms in MCF-7 cells to examine coupling of endogenous relaxin receptors to cAMP production. Overexpression of types II and IV AC had no effect on cAMP production by relaxin. However, overexpression of type V AC, which is activated by PKCζ, showed synergistic stimulation of cAMP by relaxin and forskolin.
doi:10.1196/annals.1282.044
PMCID: PMC2799245  PMID: 15956721
adenylyl cyclase; PI3K; PKCζ; relaxin; cyclic AMP; ACV; ACII
10.  The Application of DTI to Investigate White Matter Abnormalities in Schizophrenia 
Schizophrenia is a serious and disabling mental disorder that affects approximately 1% of the general population, with often devastating effects on the psychological and financial resources of the patient, family, and larger community. The etiology of schizophrenia is not known, although it likely involves several interacting biological and environmental factors that predispose an individual to schizophrenia. However, although the underlying pathology remains unknown, it has been believed that brain abnormalities would ultimately be linked to the etiology of schizophrenia. This theory was rekindled in the 1970s, when the first computer-assisted tomography (CT) study showed enlarged lateral ventricles in schizophrenia. Since that time, there have been many improvements in MR acquisition and image processing, including the introduction of positron emission tomography (PET), followed by functional MR (fMRI), and diffusion tensor imaging (DTI). These advances have led to an appreciation of the critical role that brain abnormalities play in schizophrenia. While structural MRI has proven to be useful in investigating and detecting gray matter abnormalities in schizophrenia, the investigation of white matter has proven to be more challenging as white matter appears homogeneous on conventional MRI and the fibers connecting different brain regions cannot be appreciated. With the development of DTI, we are now able to investigate white matter abnormalities in schizophrenia.
doi:10.1196/annals.1340.024
PMCID: PMC2768113  PMID: 16394153
schizophrenia; diffusion tensor imaging (DTI); frontal-temporal connections; white matter
11.  Investigating the functional role of callosal connections with dynamic causal models 
The anatomy of the corpus callosum has been described in considerable detail. Tracing studies in animals and human post-mortem experiments are currently complemented by diffusion-weighted imaging, which enables non-invasive investigations of callosal connectivity to be conducted. In contrast to the wealth of anatomical data, little is known about the principles by which inter-hemispheric integration is mediated by callosal connections. Most importantly, we lack insights into the mechanisms that determine the functional role of callosal connections in a context-dependent fashion. These mechanisms can now be disclosed by models of effective connectivity that explain neuroimaging data from paradigms which manipulate inter-hemispheric interactions. In this article, we demonstrate that Dynamic Causal Modeling (DCM), in conjunction with Bayesian model selection (BMS), is a powerful approach to disentangling the various factors that determine the functional role of callosal connections. We first review the theoretical foundations of DCM and BMS before demonstrating the application of these techniques to empirical data from a single subject.
doi:10.1196/annals.1340.008
PMCID: PMC2644452  PMID: 16394145
fMRI; DTI; DCM; effective connectivity; corpus callosum; inter-hemispheric integration
12.  Cellular and Molecular Mechanisms of Regulation of Autoantibody Production in Lupus 
The hyperactive interaction between helper T cells and autoimmune B cells in individuals predisposed to systemic lupus erythematosus (SLE) can be interrupted by induction of regulatory and suppressor T cells. Using two strategies—high dose tolerance to an immunoglobulin-derived peptide, and minigene vaccination with DNA encoding T cell epitopes presented by MHC class I molecules—our group has induced at least three types of regulatory/suppressive T cells. They include CD8+ T cells that suppress helper T cells by cytokine secretion, CD8+ T suppressors that kill B cells making anti-DNA antibodies, and peptide-binding CD4+CD25+ regulatory T cells that suppress B cells by direct cell contact. Each of these lymphocyte subsets suppresses anti-DNA antibody production and delays the onset of nephritis in BWF1 lupus-prone mice. Patients with SLE have amino acid sequences similar to those from murine anti-DNA antibodies used in these studies, and at similar locations in the VH regions of anti-DNA immunoglobulins. Therefore, strategies described here might ultimately be useful in therapy of the human disease.
doi:10.1196/annals.1361.085
PMCID: PMC2291525  PMID: 16126985
systemic lupus erythematosus; CD8+ T cells; regulatory T cells; suppressor T cells
13.  Bartonella bacilliformis GroEL; Effect on Growth of Human Vascular Endothelial Cells in Infected Co-Cultures 
Bartonella are the only bacteria known to induce angioproliferative lesions of the human vasculature and liver during infection. Previous work from our lab suggests that GroEL participates in the mitogenic response observed in HUVEC cultures supplemented with the soluble fraction of Bartonella bacilliformis. Work in this study shows that exposure to high concentrations of the fraction is actually cytotoxic for HUVECs. To analyze this phenomenon, live B. bacillformis - HUVEC co-cultures were employed to study the effect of excess bacterial GroEL on the host cell during active infection. Four B. bacilliformis strains were generated to produce varying levels of GroEL. HUVEC co-cultures with LSS100, a strain that synthesizes markedly greater quantities of GroEL relative to others, significantly accelerates apoptosis of the co-cultured HUVECs relative to other strains. Acceleration of apoptosis can be inhibited by Z-VAD-FMK, a pan-caspase inhibitor. Time course data show that at 18 h of infection, both LSS100 and control strains significantly inhibit spontaneous apoptosis of co-cultured HUVECs, as previously reported for other Bartonella species. However, by 48 h LSS100 significantly increases apoptosis of the host cell. We hypothesize that intracellular Bartonella GroEL functions as an HSP60 analog, a eukaryotic orthologue known to accelerate procaspase 3 activation by enhancing its vulnerability to upstream activator caspases. These data suggest another strategy whereby Bartonella may regulate host cell growth.
doi:10.1196/annals.1355.046
PMCID: PMC1817666  PMID: 16481529
14.  Determining Structure and Function of Steroid Dehydrogenase Enzymes by Sequence Analysis, Homology Modeling, and Rational Mutational Analysis 
The short-chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, ~300 have been characterized functionally, and the three-dimensional crystal structures of ~40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown and to determine their three-dimensional structure and mechanism of action. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30–40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including cofactor preference, catalytic residues, and substrate specificity. Human type 1 3β-hydroxysteroid dehydrogenase isomerase (3β-HSDI) has 30% sequence identity with a human UDP galactose 4-epimerase (UDPGE), a SCOR family enzyme for which an X-ray structure has been reported. Both UDPGE and 3-HSDI appear to trace their origins back to bacterial 3α,20β-HSD. Combining three-dimensional structural information and sequence data on the 3α,20β-HSD, UDPGE, and 3β-HSDI subfamilies with mutational analysis, we were able to identify the residues critical to the dehydrogenase function of 3-HSDI. We also identified the residues most probably responsible for the isomerase activity of 3β-HSDI. We test our predictions by specific mutations based on sequence analysis and our structure-based model.
doi:10.1196/annals.1336.015
PMCID: PMC1635414  PMID: 16467263
rational mutational analysis; steroid dehydrogenase; homology modeling; prostate cancer; breast cancer; prediction of ligand specificity; testosterone 3-HSDII
15.  Transgenic Rescue of SF-1-Null Mice 
Steroidogenic factor 1 (SF-1, Nr5a1, and Ad4bp) is an orphan nuclear receptor required for adrenal and gonad development and endocrine regulation. To extend our understanding of SF-1 function and the mechanisms controlling its expression, a transgenic rescue strategy was employed to locate important transcriptional control regions and to reveal functional roles of the protein. A rat yeast artificial chromosome containing Ftz-F1, the gene encoding SF-1, was used to generate mice with different transgenes that varied in size. Rat SF-1 mRNA expression was assayed to assess each transgene’s targeting ability. SF-1-deficient/transgene-positive (SF-1−/−; tg/+) “rescue” mice were then generated and the animals’ developmental and reproductive status was evaluated. The results identified differences in expression patterns and rescue abilities that provided insight into SF-1 transcriptional control and function. Comparing transgene maps and mRNA profiles placed critical transcriptional elements for pituitary and hypothalamic expression to a region 3′ to intron 4, whereas examination of rescued mice revealed that an ~153-kb region of the Ftz-F1 locus recapitulates most or all activity ascribed to the endogenous allele. A second line of rescued mice was hypomorphic, with males showing defects in androgen-dependent tissues due to abnormal Leydig cell differentiation. Histological analysis of embryonic (e14.5) and adult testes from these mice implicated SF-1 in roles that are distinct in fetal and adult Leydig cells.
doi:10.1196/annals.1336.007
PMCID: PMC1544363  PMID: 16467257
SF-1; testes; adrenal; YAC transgenic mice; steroidogenesis
16.  Phototransformations of Advanced Glycation End Products in the Human Eye Lens due to Ultraviolet A Light Irradiation 
Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP-lysine, OP-phenethylamine (a phenethylamine analogue of OP-lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP-lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP-lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.
doi:10.1196/annals.1333.021
PMCID: PMC1564128  PMID: 16037236
ascorbic acid; OP-lysine; UVA light; eye lens; glycation
17.  K2P—A Novel Cross-Link from Human Lens Protein 
We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by LC-MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure as being 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl-amino)-3-hydroxy-2, 3-dihydropyridinium, a cross-link between the _-amino groups of two lysine residues and a five-carbon atom ring. We assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 ± 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 ± 51 pmol/mg of water-soluble [WS] protein) when compared with aged normal human lens proteins (261 ± 93 pmol/mg of WISS protein or 23 ± 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross-link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.
doi:10.1196/annals.1333.023
PMCID: PMC1563990  PMID: 16037238
human lens; brunescent cataract; cross-link; advanced glycation end product; yellow chromophore; fluorophore; UVA sensitizer
18.  Functional Validation of Genes Implicated in Lymphomagenesis 
The involvement of the c-Myc transcription factor in neoplastic transformation is well documented. However, which of its numerous target genes are crucial for tumorigenesis remains a frequently contested issue. We have recently established a non-transgenic murine model for B-cell lymphoma based on neoplastic conversion of p53-null bone marrow cells by conditionally active Myc. Using this model, we have identified a number of genes whose expression levels are affected by Myc during B-lymphomagenesis. Here we discuss their possible roles in neoplastic processes and describe an experimental approach allowing in vivo validation of these roles. We demonstrate that lymphoma cells overexpressing one of the Myc targets, the interleukin-10 receptor gene, have a very strong selective advantage over low IL10R expressors. Furthermore, Mcl1, a presumptive IL10R effector, also confers selective advantages when overexpressed in Myc-transformed hematopoietic cells. Thus, both IL10R and Mcl1 might be amenable to therapeutic interventions, and new targets can be identified and validated using the selection approach.
doi:10.1196/annals.1339.047
PMCID: PMC1490324  PMID: 16382050
c-Myc; B-cell lymphoma; target genes; Mcl1; IL10
19.  Calcium Biology of the Transverse Tubules in Heart 
Ca2+ sparks in heart muscle are activated on depolarization by the influx of Ca2+ through dihydropyridine receptors in the sarcolemmal (SL) and transverse tubule (TT) membranes. The cardiac action potential is thus able to synchronize the [Ca2+]i transient as Ca2+ release is activated throughout the cell. Increases in the amount of Ca2+ within the sarcoplasmic reticulum (SR) underlie augmented Ca2+ release globally and an increase in the sensitivity of the ryanodine receptors (RyRs) to be triggered by the local [Ca2+]i. In a similar manner, phosphorylation of the RyRs by protein kinase A (PKA) increases the sensitivity of the RyRs to be activated by local [Ca2+]i. Heart failure and other cardiac diseases are associated with changes in SR Ca2+ content, phosphorylation state of the RyRs, [Ca2+]i signaling defects and arrhythmias. Additional changes in transverse tubules and nearby junctional SR may contribute to alterations in local Ca2+ signaling. Here we briefly discuss how TT organization can influence Ca2+ signaling and how changes in SR Ca2+ release triggering can influence excitation–contraction (EC) coupling. High speed imaging methods are used in combination with single cell patch clamp experiments to investigate how abnormal Ca2+ signaling may be regulated in health and disease. Three issues are examined in this presentation: (1) normal Ca2+-induced Ca2+ release and Ca2+ sparks, (2) abnormal SR Ca2+ release in disease, and (3) the triggering and propagation of waves of elevated [Ca2+]i.
doi:10.1196/annals.1341.009
PMCID: PMC1484521  PMID: 16093488
Ca2+ sparks; Ca2+ waves; ryanodine receptors; transverse tubules
20.  Modulation of Transmural Repolarization 
Ventricular myocardium in larger mammals has been shown to be comprised of three distinct cell types: epicardial, M, and endocardial. Epicardial and M cell action potentials differ from endocardial cells with respect to the morphology of phase 1. These cells possess a prominent Ito-mediated notch responsible for the “spike and dome” morphology of the epicardial and M cell response. M cells are distinguished from the other cell types in that they display a smaller IKs, but a larger late INa and INa-Ca. These ionic distinctions underlie the longer action potential duration (APD) and steeper APD-rate relationship of the M cell. Difference in the time course of repolarization of phase 1 and phase 3 are responsible for the inscription of the electrocardiographic J wave and T wave, respectively. These repolarization gradients are sensitively modulated by electrotonic communication among the three cells types, [K +]o, and the presence of drugs that either reduce or augment net repolarizing current. A reduction in net repolarizing current generally leads to a preferential prolongation of the M cell action potential, responsible for a prolongation of the QT interval and an increase in transmural dispersion of repolarization (TDR), which underlies the development of torsade de pointes arrhythmias. An increase in net repolarizing current can lead to a preferential abbreviation of the action potential of epicardium in the right ventricle (RV), and endocardium in the left ventricle (LV). These actions also lead to a TDR that manifests as the Brugada syndrome in RV and the short QT syndrome in LV.
doi:10.1196/annals.1341.028
PMCID: PMC1474840  PMID: 16093507
Brugada syndrome; cardiac heterogeneity; electrocardiogram; long QT syndrome; M cell; short QT syndrome
21.  Development of resistance to RNAi in mammalian cells 
The discovery of RNA interference (RNAi) in C. elegans and in plants has revolutionized current approaches to biology and medicine. RNAi silences genes in a sequence-specific manner through the actions of small pieces of double-stranded RNAs (siRNAs and miRNAs). RNAi has been found as a widespread natural phenomenon in eukaryotic cells and is also being used as a powerful experimental tool to explore gene function. Most importantly, it has many potential therapeutic applications. Viral gene-specific siRNAs are theoretically very promising antiviral inhibitors and have been examined in a broad range of medically important viruses. However, many RNA viruses escape RNAi-mediated suppression by counteracting the RNAi machinery through mutation of the targeted region, by encoding viral suppressors, or both. DNA viruses also counteract the RNAi machinery, preferentially using viral suppressors. Cellular factors may also contribute to RNAi resistance; ADAR1 was the first cellular factor found to be responsible for editing-mediated RNAi resistance. Because siRNAs can be used as potent small-molecule inhibitors of any cellular gene, the best way for a cell to maintain expression of essential genes for its long-term survival is to develop a program to resist the detrimental effects of RNAi.
doi:10.1196/annals.1359.019
PMCID: PMC1462965  PMID: 16394130
RNA interference; siRNA; miRNA; gene expression; viral inhibitors; gene therapies
22.  Initial Ocular Following in Humans Depends Critically on the Fourier Components of the Motion Stimulus 
Visual motion is sensed by low-level (energy-based) and high-level (feature-based) mechanisms. Our interest is in the motion detectors underlying the initial ocular following responses (OFR) that are elicited at ultrashort latencies by sudden motions of large images. OFR were elicited in humans by applying horizontal motion to vertical square-wave gratings lacking the fundamental. In the frequency domain, a pure square wave is composed of the odd harmonics—first, third, fifth, seventh, etc.—such that the third, fifth, seventh, etc., have amplitudes that are one-third, one-fifth, one-seventh, etc., that of the first, and the missing fundamental stimulus lacks the first harmonic. Motion consisted of successive quarter-wavelength steps, so the features and 4n+1 harmonics (where n = integer) shifted forward, whereas the 4n-1 harmonics—including the strongest Fourier component (the third harmonic)—shifted backward (spatial aliasing). Thus, the net Fourier energy and the non-Fourier features moved in opposite directions. Initial OFR, recorded with the search coil technique, had minimum latencies of 60 to 70 ms and were always in the direction of the third harmonic, for example, leftward steps resulted in right-ward OFR. Thus, the earliest OFR were strongly dependent on the motion of the major Fourier component, consistent with mediation by oriented spatiotemporal visual filters as in the well-known energy model of motion detection. Introducing interstimulus intervals of 10 to 100 ms (during which the screen was uniform gray) reversed the initial direction of tracking, consistent with extensive neurophysiological and psychophysical data suggesting that the visual input to the motion detectors has a biphasic temporal impulse response.
doi:10.1196/annals.1325.025
PMCID: PMC1383627  PMID: 15826980
visual motion; energy-based mechanisms; biphasic temporal impulse response; missing fundamental
23.  Short-Latency Disparity Vergence in Humans: Evidence for Early Spatial Filtering 
Our study was concerned with the disparity detectors underlying the initial disparity vergence responses (DVRs) that are elicited at ultrashort latencies by binocular disparities applied to large images. DVRs were elicited in humans by applying horizontal disparity to vertical square-wave gratings lacking the fundamental (termed here, the “missing fundamental”). In the frequency domain, a pure square wave is composed of odd harmonics—first, third, fifth, seventh, etc.—such that the third, fifth, seventh, etc., have amplitudes that are one-third, one-fifth, one-seventh, etc., that of the first, and the missing fundamental lacks the first harmonic. The patterns seen by the two eyes have a phase difference of one-quarter wavelength, so the disparity of the features and 4n + 1 harmonics (where n = integer) has one sign (crossed or uncrossed), whereas the 4n - 1 harmonics—including the strongest Fourier component (the third harmonic)—has the opposite sign (uncrossed or crossed): spatial aliasing. The earliest DVRs, recorded with the search-coil technique, had minimum latencies of 70 to 80 ms and were generally in the direction of the third harmonic, that is, uncrossed disparities resulted in convergent eye movements. In other experiments on the DVRs, one eye saw a missing fundamental and the other saw a pure sine wave with the contrast and wavelength of the third harmonic but differing in phase by one-quarter wavelength. This resulted in short-latency vergence in accordance with matching of the third harmonic. These data all indicate the importance of the Fourier components, consistent with early spatial filtering prior to binocular matching.
doi:10.1196/annals.1325.024
PMCID: PMC1369053  PMID: 15826979
missing fundamental; binocular disparity; vergence eye movements

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