Population and molecular evolution studies on the Melanocortin 1 receptor locus implicate its role in human pigmentation variation

Human pigmentation is a complex trait with the observed variation caused by the varied production of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) by the melanocytes. The melanocortin 1 receptor (MC1R), a G protein-coupled receptor expressed in the melanocytes, is a regulator eu- and phaeomelanin synthesis, and MC1R mutations causing skin and coat color changes are known in many mammals. To understand the role of MC1R in human pigmentation variation, I have sequenced the MC1R gene in 121 individuals sampled from world populations. In addition, I have sequenced the MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon to study the evolution of MC1R and to infer the ancestral human MC1R sequence. The ancestral MC1R sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. To further evaluate the role of MC1R variants in human pigmentation variation, I have combined these molecular evolution and population studies with functional assays on MC1R variants and primate MC1Rs. ^

Linkage and association of candidate obesity genes in Mexican-Americans from Starr County, Texas

Obesity and related chronic diseases represent a tremendous public health burden among Mexican Americans, a young and rapidly-expanding population. This study investigated the impact of variation within eight candidate obesity genes, which include leptin (LEP), leptin receptor (LEPR), neuropeptide Y (NPY), NPYY1 receptor (NPYY1), glucagon-like peptide-1 (GLP-1), GLP-1 receptor (GLP1R), beta-3 adrenergic receptor (β3AR), and uncoupling protein (UCP1), on variation in human obesity status and/or quantitative traits related to obesity in Mexican Americans from Starr County, Texas. The Trp64Arg polymorphism within β3AR was typed in 820 random individuals and 240 pedigrees (N = 2,044). The Arg allele frequency was significantly greater in obese versus non-obese individuals (0.20 versus 0. 15, respectively). In addition, within the random sample, the Arg allele was associated with significantly greater body weight (p = 0.031) and body mass index (BMI, p = 0.008) than the Trp allele. In the family sample, the Trp64Arg locus was also linked to percent fat (p = 0.045) but not to body weight or BMI. No linkage between obesity, diabetes, hypertension, or gallbladder disease and the Trp64Arg mutation was observed in families using affected sib pair linkage analysis or the transmission disequilibrium test. Microsatellite markers proximate to the remaining seven genes were typed in 302 individuals from 59 families. Sib pair linkage analysis provided evidence for linkage between obesity and NPY within affected sibling pairs (p = 0.042; n = 170 pairs). NPY was also linked to weight (p = 0.020), abdominal circumference (p = 0.031), hip circumference (p = 0.012), DBP (p ≤ 0.005), and a composite measure of body mass/fat (p ≤ 0.048) in all sibling pairs (n = 545 pairs). Additionally, LEP was linked to waist/hip ratio (p ≤ 0.009), total cholesterol (p ≤ 0.030), and HDL cholesterol (p ≤ 0.026), and LEPR was linked to fasting blood glucose (p ≤ 0.018) and DBP (p ≤ 0.003). Subsequent to the linkage analyses, the NPY gene was sequenced and eight variant sites identified. Two variant sites (-880I/D and 69I/D) were typed in a random sample of 914 individuals. The 880I/D variant was significantly associated with waist/hip ratio (p = 0.035) in the entire sample (N = 914) and with BMI (p = 0. 031), abdominal circumference (p = 0.044), and waist/hip ratio (p = 0.041) in a non-obese subsample (BW < 30 kg/m2, n = 594). The 69I/D variant was a rare mutation observed in only one pedigree and was not associated with obesity or body size/mass within this pedigree. Results of this study indicate that variation at or near β3AR, LEP, LEPR, and NPY may exert effects which increase obesity susceptibility and influence obesity-related measures in this population. ^

The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia.

Growth-restricted fetuses are at risk for a variety of lifelong medical conditions. Preeclampsia, a life-threatening hypertensive disorder of pregnancy, is associated with fetuses who suffer from intrauterine growth restriction (IUGR). Recently, emerging evidence indicates that preeclamptic women harbor AT(1) receptor agonistic autoantibodies (AT(1)-AAs) that contribute to the disease features. However, the exact role of AT(1)-AAs in IUGR and the underlying mechanisms have not been identified. We report that these autoantibodies are present in the cord blood of women with preeclampsia and retain the ability to activate AT(1) receptors. Using an autoantibody-induced animal model of preeclampsia, we show that AT(1)-AAs cross the mouse placenta, enter fetal circulation, and lead to small fetuses with organ growth retardation. AT(1)-AAs also induce apoptosis in the placentas of pregnant mice, human villous explants, and human trophoblast cells. Finally, autoantibody-induced IUGR and placental apoptosis are diminished by either losartan or an autoantibody-neutralizing peptide. Thus, these studies identify AT(1)-AA as a novel causative factor of preeclampsia-associated IUGR and offer two possible underlying mechanisms: a direct detrimental effect on fetal development by crossing the placenta and entering fetal circulation, and indirectly through AT(1)-AA-induced placental damage. Our findings highlight AT(1)-AAs as important therapeutic targets.

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes.

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

TIME DEPENDENT ALTERATIONS IN CENTRAL NERVOUS SYSTEM DOPAMINE RECEPTORS INDUCED BY DOPAMINE AGONIST TREATMENT

Levodopa, the precursor of dopamine, is currently the drug of choice in the treatment of Parkinson's disease. Recently, two direct dopamine agonists, bromocriptine and pergolide, have been tested for the treatment of Parkinson's disease because of reduced side effects compared to levodopa. Few studies have evaluated the effects of long-term treatment of dopamine agonists on dopamine receptor regulation in the central nervous system. Thus, the purpose of this study was to determine whether chronic dopamine agonist treatment produces a down-regulation of striatal dopamine receptor function and to compare the results of the two classes of dopaminergic drugs.^ Levodopa with carbidopa, a peripheral decarboxylase inhibitor, was administered orally to rats whereas bromocriptine and pergolide were injected intraperitoneally once daily. Several neurochemical parameters were examined from 1 to 28 days.^ Levodopa minimally decreased striatal D-1 receptor activity but increased the number of striatal D-2 binding sites. Levodopa increased the V(,max) of tyrosine hydroxylase (TH) in all brain regions tested. Protein blot analysis of striatal TH indicated a significant increase in the amount of TH present. Dopamine-beta-hydroxylase (DBH) activity was markedly decreased in all brain regions studied and mixing experiments of control and drug-treated cortices did not show the presence of an increased level of endogenous inhibitors.^ Bromocriptine treatment decreased the number of D-2 binding sites. Striatal TH activity was decreased and protein blot analysis indicated no change in TH quantity. The specificity of bromocriptine for striatal TH suggested that bromocriptine preferentially interacts with dopamine autoreceptors.^ Combination levodopa-bromocriptine was administered for 12 days. There was a decrease in both D-1 receptor activity and D-2 binding sites, and a decrease in brain HVA levels suggesting a postsynaptic receptor action. Pergolide produced identical results to the combination levodopa-bromocriptine studies.^ In conclusion, combination levodopa-bromocriptine and pergolide treatments exhibited the expected down-regulation of dopamine receptor activity. In contrast, levodopa appeared to up-regulate dopamine receptor activity. Thus, these data may help to explain, on a biochemical basis, the decrease in the levodopa-induced side effects noted with combination levodopa-bromocriptine or pergolide therapies in the treatment of Parkinson's disease. ^

The role of AT1 receptor autoantibodies in the pathophysiology of preeclampsia

Preeclampsia is a disease that affects 3–5% of all pregnancies. The cause is unknown and there is currently no treatment. The disease poses significant health risks to both the mother and the fetus. To date, research on the topic has not produced a convincing cause for the development of the hallmark symptoms of preeclampsia. The hypothesis of an agonistic autoimmune response to the AT1 receptor is presented. Immunoglobulin fractions from normotensive and preeclampsia patients were prepared for experimental tests. Model systems were tested in three categories to determine if AT 1 receptor specific activation and receptor-ligand interaction was caused by a suspected autoantibody. Activation was found in rat neonatal cardiornyocytes that caused an increased contraction rate. This activity was found in preeclampsia patients, absent in normotensive patients. The activation was antagonized by losartan, an AT1 receptor antagonist, and by epitope peptide competition of the receptor-ligand type interaction. This epitope was the 7 amino acid peptide fragment, AFHYESQ, a sequence present in the second extracellular loop of the AT1 receptor. The patterns of AT1 receptor activation were also found in a human trophoblast cell line, HTR8, with an effect on Pai-1 secretion, a factor that plays a role in preventing hypercoagulation. In human mesangial cells, the AT1 receptor autoantibody present in the immunoglobulin fraction from preeclampsia patients was found to stimulate the secretion of Pai-1, and IL-6, a factor that plays a role in the activation of an inflammatory response. This activity was found in samples from preeclampsia patients, but absent in normotensive patients. Tests including losartan, AFHYESQ, and a non-competitive peptide demonstrated that the secretion of Pai-1 and IL-6 met the criteria for AT1 receptor activation by the suspected agonistic autoantibody. These three model systems address relevant pathophysiology for preeclampsia patients, including increased cardiac output, abnormal placentation, and renal damage. The AT1 receptor agonistic autoantibody is potentially a key player in the development of the pathology and symptoms of preeclampsia. ^

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^

Genes to blood pressure: The effects of variation in genes of the renin-angiotensin system on the hormonal and renal control of blood pressure

Objective. Essential hypertension affects 25% of the US adult population and is a leading contributor to morbidity and mortality. Because BP is a multifactorial phenotype that resists simple genetic analysis, intermediate phenotypes within the complex network of BP regulatory systems may be more accessible to genetic dissection. The Renin-Angiotensin System (RAS) is known to influence intermediate and long-term blood pressure regulation through alterations in vascular tone and renal sodium and fluid resorption. This dissertation examines associations between renin (REN), angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) gene variation and interindividual differences in plasma hormone levels, renal hemodynamics, and BP homeostasis.^ Methods. A total of 150 unrelated men and 150 unrelated women, between 20.0 and 49.9 years of age and free of acute or chronic illness except for a history of hypertension (11 men and 7 women, all off medications), were studied after one week on a controlled sodium diet. RAS plasma hormone levels, renal hemodynamics and BP were determined prior to and during angiotensin II (Ang II) infusion. Individuals were genotyped by PCR for a variable number tandem repeat (VNTR) polymorphism in REN, and for the following restriction fragment length polymorphisms (RFLP): AGT M235T, ACE I/D, and AT1 A1166C. Associations between clinical measurements and allelic variation were examined using multiple linear regression statistical models.^ Results. Women homozygous for the AT1 1166C allele demonstrated higher intracellular levels of sodium (p = 0.044). Men homozygous for the AGT T235 allele demonstrated a blunted decrement in renal plasma flow in response to Ang II infusion (p = 0.0002). There were no significant associations between RAS gene variation and interindividual variation in RAS plasma hormone levels or BP.^ Conclusions. Rather than identifying new BP controlling genes or alleles, the study paradigm employed in this thesis (i.e., measured genes, controlled environments and interventions) may provide mechanistic insight into how candidate genes affect BP homeostasis. ^

Mechanisms of TGF-beta1-induced neuronal plasticity in Aplysia

Transforming growth factor beta-1 (TGF-β1) is a cytokine and neurotrophic factor whose neuromodulatory effects in Aplysia californica were recently described. Previous results demonstrated that TGF-β1 induces long-term increases in the efficacy of sensorimotor synapses, a neural correlate of sensitization of the defensive tail withdrawal reflex. These results provided the first evidence that a neurotrophic factor regulates neuronal plasticity associated with a simple form of learning in Aplysia, and raised many questions regarding the nature of the modulation. No homologs of TGF-β had previously been identified in Aplysia, and thus, it was not known whether components of TGF-β1 signaling pathways were present in Aplysia. Furthermore, the signaling mechanisms engaged by TGF-β1 had not been identified, and it was not known whether TGF-β1 regulated other aspects of neuronal function.^ The present investigation into the actions of TGF-β1 was initiated by examining the distribution of the type II TGF-β1 receptor, the ligand binding receptor. The receptor was widely distributed in the CNS and most neurons exhibited somatic and neuritic immunoreactivity. In addition, the ability of TGF-β1 to activate the cAMP/PKA and MAPK pathways, known to regulate several important aspects of neuronal function, was examined. TGF-β1 acutely decreased cAMP levels in sensory neurons, activated MAPK and triggered translocation of MAPK to the nucleus. MAPK activation was critical for both short- and long-term regulation of neuronal function by TGF-β1. TGF-β1 acutely decreased synaptic depression induced by low frequency stimuli in a MAPK-dependent manner. This regulation may result, at least in part, from the modulation of synapsin, a major peripheral synaptic vesicle protein. TGF-β1 stimulated MAPK-dependent phosphorylation of synapsin, a process believed to regulate synaptic vesicle mobilization from reserve to readily-releasable pools of neurotransmitter. In addition to its acute effect on synaptic efficacy, TGF-β1 also induced long-term increases in sensory neuron excitability. Whereas transient exposure to TGF-β1 was not sufficient to drive short-or long-term changes in excitability, prolonged exposure to TGF-β1 induced long-term changes in excitability that depended on MAPK. The results of these studies represent significant progress toward an understanding of the role of TGF-β1 in neuronal plasticity. ^

Adenosine mediated lung mast cell degranulation in the mouse

Adenosine has been implicated to play a role in inflammatory processes associated with asthma. Most notable is adenosine's ability to potentiate mediator release from mast cells. Mast cells are bone marrow derived inflammatory cells that can release mediators that have both immediate and chronic effects on airway constriction and inflammation. Most physiological roles of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B and A 3. The mechanisms by which adenosine can influence the release of mediators from lung tissue mast cells is not understood due to lack of in vivo models. Mice deficient in the enzyme adenosine deaminase (ADA) have been generated. ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice. ADA-deficient mice develop features seen in asthmatics, including lung eosinophilia and mucus hypersecretion. In addition, lung tissue mast cell degranulation was associated with elevated adenosine in ADA-deficient lungs and can be prevented by ADA enzyme therapy. We established primary murine lung mast cell cultures, and used real time RT-PCR and immunofluorescence to demonstrate that A 2A, A2B and A3 receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists and A3 receptor deficient (A3−/−) mast cells suggested that activation of A3 receptors could induce mast cell mediator release in vitro. Furthermore, this mediator release was associated with increases in intracellular Ca++ that appeared to be mediated through a Gi and PI3K pathway. In addition, nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild type mice while having no effect in A3−/− mice. These results demonstrate that the A3 receptor plays an important role in adenosine mediated murine lung mast cell degranulation. Therefore, the A3 adenosine receptor and its signaling pathways may represent novel therapeutic targets for the treatment and prevention of asthma. ^

$\beta\sb2$-adrenergic receptor desensitization, internalization and phosphorylation in response to full and partial agonists

The objective of this study is to test the hypothesis that partial agonists produce less desensitization because they generate less of the active conformation of the $\beta\sb2$-adrenergic receptor ($\beta$AR) (R*) and in turn cause less $\beta$AR phosphorylation by beta adrenergic receptor kinase ($\beta$ARK) and less $\beta$AR internalization. In the present work, rates of desensitization, internalization, and phosphorylation caused by a series of $\beta$AR agonists were correlated with a quantitative measure, defined as coupling efficiency, of agonist-dependent $\beta$AR activation of adenylyl cyclase. These studies were preformed in HEK-293 cells overexpressing the $\beta$AR with hemagglutinin (HA) and 6-histidine (6HIS) epitopes introduced into the N- and C-termini respectively. Agonists chosen provided a 95-fold range of coupling efficiencies, and, relative to epinephrine, the best agonist, (100%) were fenoterol (42%), albuterol (4.9%), dobutamine (2.5%) and ephedrine (1.1%). At concentrations of these agonists yielding $>$90% receptor occupancy, the rate and extent of the rapid phase (0-30 min) of agonist induced desensitization of adenylyl cyclase followed the same order as coupling efficiency, that is, epinephrine $\ge$ fitnoterol $>$ albuterol $>$ dobutamine $>$ ephedrine. The rate of internalization, measured by a loss of surface receptors during desensitization, with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like desensitization and internalization, $\beta$AR phosphorylation exhibited a dependency on agonist strength. The two strongest agonists epinephrine and fenoterol provoked 11 to 13 fold increases in the level of $\beta$AR phosphorylation after just 1 min, whereas the weakest agonists dobutamine and ephedrine caused only 3 to 4 fold increases in phosphorylation. With longer treatment times, the level of $\beta$AR phosphorylation declined with the strong agonists, but progressively increased with the weaker partial agonists. The major conclusion drawn from this study is that the occupancy-dependent rate of receptor phosphorylation increases with agonist coupling efficiencies and that this is sufficient to explain the desensitization, internalization, and phosphorylation data obtained.^ The mechanism of activation and desensitization by the partial $\beta$AR agonist salmeterol was also examined in this study. This drug is extremely hydrophobic and its study presents possibly unique problems. To determine whether salmeterol induces desensitization of the $\beta$AR its action has been studied using our system. Employing the use of reversible antagonists it was found that salmeterol, which has an estimated coupling efficiency near that of albuterol caused $\beta$AR desensitization. This desensitization was much reduced relative to epinephrine. Consistent with its coupling efficiency, it was found to be similar to albuterol in its ability to induce internalization and phosphorylation of the $\beta$AR. (Abstract shortened by UMI.) ^