LabAutomation 2001. Part 4: Mass spectroscopy as sensitive detector, protein function in disease, and the soul of a robot.
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Artificial Intelligence↗
Biomedical subjects
R A Felder
Publications and source records attributed to R A Felder.
Is lab automation right for your lab?
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Automation↗
Dopamine receptors in hypertension.
There is increased awareness of the role of dopamine in cardiovascular function, renal function and systemic blood pressure regulation. Growing evidence indicates that each of the five dopamine receptor subtypes participates in the regulation of blood pressure by mechanisms distinct for that particular subtype. Some dopamine receptors regulate blood pressure by influencing the central and peripheral nervous system, while others influence renal function and release of renin, aldosterone and vasopressin. This review summarizes the physiology and pathophysiology of the peripheral dopaminergic system and our current understanding of the role of individual dopamine receptors in the pathophysiology of human essential hypertension.
Blood Pressure↗
Robotic automation performs a nested RT-PCR analysis for HCV without introducing sample contamination.
The Polymerase Chain Reaction (PCR) is a popular method to amplify and detect specific RNA and DNA sequences. To obtain maximum performance of PCR, it is best performed by highly skilled technologists because of the complexity of the assay and the potential for laboratory contamination from the amplification products produced. We chose to automate this nested RT-PCR for hepatitis C assay to significantly reduce the need for manual pipetting while preserving the excellent non-contamination performance of the corresponding manual test. A three axis cartesian robotic pipetting station was equipped to perform RT-PCR using an on-board automated thermal cycling device. 104 sera were analyzed using this modified pipetting station and we found a very close agreement (100% sensitivity and 98% specificity) with results previously obtained by corresponding manual RT-PCR analysis. This study demonstrated a user-programmed robotic pipetting system could successfully automate a complex PCR assay without contamination. Our results suggest that use of robotic pipetting station can provide cost efficient alternative to performance of molecular diagnostic assays while demonstrating minimal inter sample contamination.
Electrophoresis, Agar Gel↗
Renal dopamine and sodium homeostasis.
During the past decade, it has become evident that dopamine plays an important role in the regulation of fluid and electrolyte balance and blood pressure. Dopamine exerts its actions through two families of dopamine receptors, designated D1-like and D2-like, which are identical in the brain and in peripheral tissues. The two D1-like receptors--D1 and D5 receptors--expressed in mammals are linked to stimulation of adenylyl cyclase. The three D2-like receptors--D2, D3, and D4,--are linked to inhibition of adenylyl cyclase. Dopamine affects fluid and electrolyte balance by regulation of renal excretion of electrolytes and water through actions on renal hemodynamics and tubular epithelial transport and by modulation of the secretion and/or action of vasopressin, renin, aldosterone, catecholamines, and endothelin B receptors (ETB) receptors. It also affects fluid and sodium intake by way of "appetite" centers in the brain and alterations of gastrointestinal tract transport. The production of dopamine in neural and non-neural tissues and the presence of receptors in these tissues suggest that dopamine can act in an autocrine or paracrine fashion. This renal autocrine-paracrine function, which becomes most evident during extracellular fluid volume expansion, is lost in essential hypertension and in some animal models of genetic hypertension. This deficit may be caused by abnormalities in renal dopamine production and polymorphisms or abnormal post-translational modification and regulation of dopamine receptor subtypes.
Adenylyl Cyclases↗
Simulation of robotic courier deliveries in hospital distribution services.
Flexible automation in the form of robotic couriers holds the potential for decreasing operating costs while improving delivery performance in hospital delivery systems. This paper discusses the use of simulation modeling to analyze the costs, benefits, and performance tradeoffs related to the installation and use of a fleet of robotic couriers within hospital facilities. The results of this study enable a better understanding of the delivery and transportation requirements of hospitals. Specifically, we examine how a fleet of robotic couriers can meet the performance requirements of the system while maintaining cost efficiency. We show that for clinical laboratory and pharmaceutical deliveries a fleet of six robotic couriers can achieve significant performance gains in terms of turn-around time and delivery variability over the current system of three human couriers per shift or 13 FTEs. Specifically, the simulation results indicate that using robotic couriers to perform both clinical laboratory and pharmaceutical deliveries would result in a 34% decrease in turn-around time, and a 38% decrease in delivery variability. In addition, a break-even analysis indicated that a positive net present value occurs if nine or more FTEs are eliminated with a resulting ROI of 12%. This analysis demonstrates that simulation can be a valuable tool for examining health care distribution services and indicates that a robotic courier system may yield significant benefits over a traditional courier system in this application.
Computer Simulation↗
D1 dopamine receptor signalling defect in spontaneous hypertension.
Dopamine modulates cardiovascular function by actions in the central and peripheral nervous system, by altering the secretion/release of prolactin, pro-opiomelanocortin, vasopressin, aldosterone, and renin, and by directly affecting renal function. Dopamine produced by the renal proximal tubule exerts an autocrine/paracrine action via two classes of dopamine receptors, D1-like (D1 and D5) and D2-like (D2, D3, and D4), that are differentially expressed along the nephron. The autocrine/paracrine function of dopamine, manifested by tubular rather than by haemodynamic mechanisms, becomes most evident during extracellular fluid volume expansion. This renal autocrine/paracrine function is lost in essential hypertension and in some animal models of genetic hypertension. The molecular basis for the dopaminergic dysfunction in hypertension may involve an abnormal post-translational modification of dopamine receptors.
Animals↗
The effect of docarpamine, a dopamine pro-drug, on blood pressure and catecholamine levels in spontaneously hypertensive rats.
We studied the effects of bolus intravenous injection of the dopamine prodrug, docarpamine (200 microg/kg), on mean arterial pressure (MAP) and heart rate (HR) in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). In WKY rats (n=18), MAP and HR increased 5 min after docarpamine and then returned to baseline levels within 15 min. In contrast, in SHRs (n=15), MAP and HR gradually decreased, reaching a nadir 20 min after injection. Five min after docarpamine, plasma dopamine and 3,4-dihydroxy phenyl acetic (DOPAC) levels increased in both WKY rats (n=5) and SHRs (n=5). The docarpamine-induced changes in MAP and HR in both rat strains (n=5/strain) were blocked by the D1-like antagonist, SCH23390. alpha-Adrenergic (n=4) and vasopressin V1 (n=3) receptor blockade also abrogated the effects of docarpamine in WKY rats. We conclude that docarpamine differentially affects MAP and HR in WKY and SHRs. In SHRs, the depressor and bradycardiac effects of docarpamine are mediated by D1-like receptors. In WKY rats, the pressor and tachycardiac responses are caused by an interaction among D1-like, alpha-adrenergic, and V1 receptors.
Adrenergic alpha-Antagonists↗
Combinations of variations in multiple genes are associated with hypertension.
The genetic analysis of hypertension has revealed complex and inconsistent results, making it difficult to draw clear conclusions regarding the impact of specific genes on blood pressure regulation in diverse human populations. Some of the confusion from previous studies is probably due to undetected gene-gene interactions. Instead of focusing on the effects of single genes on hypertension, we examined the effects of interactions of alleles at 4 candidate loci. Three of the loci are in the renin-angiotensin-system, angiotensinogen, ACE, and angiotensin II type 1 receptor, and they have been associated with hypertension in at least 1 previous study. The fourth locus studied is a previously undescribed locus, named FJ. In total, 7 polymorphic sites at these loci were analyzed for their association with hypertension in 51 normotensive and 126 hypertensive age-matched individuals. There were no significant differences between the 2 phenotypic classes with respect to either allele or genotype frequencies. However, when we tested for nonallelic associations (linkage disequilibrium), we found that of the 120 multilocus comparisons, 16 deviated significantly from random in the hypertensive class, but there were no significant deviations in the normotensive group. These findings suggest that genetic interactions between multiple loci rather than variants of a single gene underlie the genetic basis of hypertension in our study subjects. We hypothesize that such interactions may account for the inconsistent findings in previous studies because, unlike our study, prior studies almost always examined single-locus effects and did not consider the effects of variation at other potentially interacting loci.
Adolescent↗
Renal protein phosphatase 2A activity and spontaneous hypertension in rats.
The impaired renal paracrine function of dopamine in spontaneously hypertensive rats (SHR) is caused by hyperphosphorylation and desensitization of the renal D(1) dopamine receptor. Protein phosphatase 2A (PP(2A)) is critical in the regulation of G-protein-coupled receptor function. To determine whether PP(2A) expression and activity in the kidney are differentially regulated in genetic hypertension, we examined the effects of a D(1)-like agonist, fenoldopam, in renal cortical tubules and immortalized renal proximal tubule cells from normotensive Wistar-Kyoto rats (WKY) and SHR. In cortical tubules and immortalized proximal tubule cells, PP(2A) expression and activities were greater in cytosol than in membrane fractions in both WKY and SHR. Although PP(2A) expressions were similar in WKY and SHR, basal PP(2A) activity was greater in immortalized proximal tubule cells of SHR than WKY. In immortalized proximal tubule cells of WKY, fenoldopam increased membrane PP(2A) activity and expression of the regulatory subunit PP(2A)-B56alpha, effects that were blocked by the D(1)-like antagonist SCH23390. Fenoldopam had no effect on cytosolic PP(2A) activity but decreased PP(2A)-B56alpha expression. In contrast, in immortalized proximal tubule cells of SHR, fenoldopam decreased PP(2A) activity in both membranes and cytosol but predominantly in the membrane fraction, without affecting PP(2A)-B56alpha expression; this effect was blocked by the D(1)-like antagonist SCH23390. We conclude that renal PP(2A) activity and expression are differentially regulated in WKY and SHR by D(1)-like receptors. A failure of D(1)-like agonists to increase PP(2A) activity in proximal tubule membranes may be a cause of the increased phosphorylation of the D(1) receptor in the SHR.
Animals↗
Modular robotic workcell for coagulation analysis.
BACKGROUND: Total laboratory automation (TLA) has been shown to increase laboratory efficiency and quality. However, modular automation is smaller, requires less initial capital, and requires less planning than TLA. We engineered and performed clinical trials on a modular robotic preanalytical workcell for coagulation analysis. METHODS: Timing studies were used to quantify the efficiency of the manual processes and to identify areas in the processing of coagulation specimens where bottlenecks and long waiting periods were encountered. We then designed our modular robotic system to eliminate these bottlenecks. Our robotic modular workcell was engineered to allow a choice of specimen introduction manually, by conveyor, or by mobile robot. Additional timing studies were performed during clinical trials of the robotic system. RESULTS: Prior to automation, the time required for preanalytical processing time was 18-107 min; after automation, it was 45-50 min. Additional improvements in workcell efficiency could be realized when high quality, prelabeled specimens were introduced into the system. CONCLUSION: Compared with manual methods, modular automation provides more predictable variation in specimen processing.
Blood Chemical Analysis↗
Selective inhibition of the renal dopamine subtype D1A receptor induces antinatriuresis in conscious rats.
Both dopamine D1-like (D1A and D1B) and D2-like (D2, D3, and D4) receptor subfamilies are present in the kidney. Blockade of the intrarenal D1-like receptor family is associated with natriuresis and diuresis. Because the D1A and D1B receptor subtypes are not distinguishable by currently available dopaminergic agents, their functional role remains undefined. In the present study, the effect of selective inhibition of the renal D1A receptor with phosphorothioated antisense oligodeoxynucleotide (AS-ODN) was investigated in conscious uninephrectomized rats. After renal interstitial administration of Texas red-labeled D1A receptor AS-ODN, intense fluorescent signal was localized in the renal tubular epithelium and vasculature. In rats on normal salt intake, AS-ODN injected interstitially into the kidney reduced daily urinary sodium excretion (1.4+/-0.04 versus 0.8+/-0.2 mEq/d, n=5, P<0.05) and urine output (16.9+/-3.8 versus 12.5+/-3.6 mL/d, n=5, P<0.05). In rats on high sodium intake, continuous renal interstitial administration of D1A receptor AS-ODN transiently decreased daily urinary sodium excretion (5.4+/-0.5 versus 4.2+/-0.3 mEq/d, n=7, P<0.01) and urine output (27.6+/-4.5 versus 18.1+/-1.8 mL/d, n=7, P<0.01). Neither vehicle nor sense oligodeoxynucleotide had significant effects. Systolic blood pressure remained unchanged. The renal D1A receptor protein was significantly decreased by 35% and 46% at the end of the study in AS-ODN-treated rats on normal and high salt intake, respectively, whereas the D1B receptor and beta-actin were not affected. These results provide the first direct evidence that the renal D1A receptor subtype plays an important role in the control of sodium excretion.
Animals↗
Dopamine-1 receptor coupling defect in renal proximal tubule cells in hypertension.
The ability of the dopamine-1 (D1)-like receptor to stimulate adenylyl cyclase (AC) and phospholipase C (PLC), inhibit sodium transport in the renal proximal tubule (RPT), and produce natriuresis is attenuated in several rat models of hypertension. Since the inhibitory effect of D1-like receptors on RPT sodium transport is also reduced in some patients with essential hypertension, we measured D1-like receptor coupling to AC and PLC in cultures of human RPT cells from normotensive (NT) and hypertensive (HT) subjects. Basal cAMP concentrations were the same in NT (n=6) and HT (n=4). However, the D1-like receptor agonist fenoldopam increased cAMP production to a greater extent in NT (maximum response=67+/-1%) than in HT (maximum response=17+/-5%), with a potency ratio of 105. Dopamine also increased cAMP production to a greater extent in NT (32+/-3%) than in HT (14+/-3%). The fenoldopam-mediated increase in cAMP production was blocked by SCH23390 (a D1-like receptor antagonist) and by antisense D1 oligonucleotides in both HT and NT, indicating action at the D1 receptor. The stimulatory effects of forskolin and parathyroid hormone-related protein of cAMP accumulation were not statistically different in NT and HT, indicating receptor specificity and an intact G-protein/AC pathway. The fenoldopam-stimulated PLC activity was not impaired in HT, and the primary sequence and expression of the D1 receptor were the same in NT and HT. However, D1 receptor serine phosphorylation in the basal state was greater in HT than in NT and was not responsive to fenoldopam stimulation in HT. These studies demonstrate the expression of D1 receptors in human RPT cells in culture. The uncoupling of the D1 receptor in both rats (previously described) and humans (described here) suggests that this mechanism may be involved in the pathogenesis of hypertension; the uncoupling may be due to ligand-independent phosphorylation of the D1 receptor in hypertension.
Aged↗
Role of dopamine in the pathogenesis of hypertension.
1. Dopamine, via different dopamine receptor subtypes, regulates cardiovascular functions by actions on the central and peripheral nervous systems, vascular smooth muscle, the heart and the kidney. The dopaminergic system in the central nervous system (CNS) may participate in the regulation of systemic blood pressure. 2. Dopamine 'D2-like' (D2, D3 and D4) receptors, rather than 'D1-like' (D1 and D5) receptors, are involved in the CNS regulation of blood pressure; post-synaptic D2-like receptors increase blood pressure, while presynaptic D2-like receptors (the predominant action) produce the opposite effect. 3. Outside the CNS, dopamine may regulate blood pressure via pressure controls that act with intermediate rapidity (e.g. stress relaxation, arginine vasopressin and renin-angiotensin vasoconstriction), as well as those systems related to the long-term control of body fluid volume. 4. Dopamine D1- and D2-like receptors have been described in resistance vessels, such as the renal, mesenteric, coronary, pulmonary and cerebral arteries. The ability of D1-like receptors to inhibit renal smooth muscle hypertrophy indicates their importance in longer-term regulation of blood pressure. 5. Aberrant dopaminergic regulation of aldosterone secretion, via D2-like receptors, has been reported to be involved in some forms of hyperaldosteronism and hypertension. Some forms of hypertension may also be caused by an aberrant renal dopaminergic system. Abnormalities of three aspects of the renal dopaminergic system may lead to hypertension: (i) renal production of dopamine; (ii) transduction of the renal vascular dopamine signal; and (iii) transduction of the renal tubular dopamine signal. 6. Thus, increased blood pressure occurs after either blockade of D1-like receptors or of dopamine production in rats or disruption of the D1 receptor or the D3 receptor gene in mice.
Animals↗
Reading the future: the increased relevance of laboratory medicine in the next century.
Through intelligent process control and data management, the laboratory may become the most frequently used--and the most important--source of diagnostic information in medicine. The central laboratory of the future is destined to become an esoteric testing center, whereas routine testing--administered at the patient bedside or at home--will become more economical. Point-of-care testing will soon become the most profitable way to provide laboratory services. Novel phlebotomy techniques and noninvasive tests may allow some diagnostic testing to be done through automated robotic companions that serve homebound patients or the elderly.
Autoanalysis↗
Disruption of the dopamine D3 receptor gene produces renin-dependent hypertension.
Since dopamine receptors are important in the regulation of renal and cardiovascular function, we studied the cardiovascular consequences of the disruption of the D3 receptor, a member of the family of D2-like receptors, expressed in renal proximal tubules and juxtaglomerular cells. Systolic and diastolic blood pressures were higher (approximately 20 mmHg) in heterozygous and homozygous than in wild-type mice. An acute saline load increased urine flow rate and sodium excretion to a similar extent in wild-type and heterozygous mice but the increase was attenuated in homozygous mice. Renal renin activity was much greater in homozygous than in wild-type mice; values for heterozygous mice were intermediate. Blockade of angiotensin II subtype-1 receptors decreased systolic blood pressure for a longer duration in mutant than in wild-type mice. Thus, disruption of the D3 receptor increases renal renin production and produces renal sodium retention and renin-dependent hypertension.
Angiotensin I↗
Modular workcells: modern methods for laboratory automation.
Laboratory automation is beginning to become an indispensable survival tool for laboratories facing difficult market competition. However, estimates suggest that only 8% of laboratories will be able to afford total laboratory automation systems. Therefore, automation vendors have developed alternative hardware configurations called 'modular automation', to fit the smaller laboratory. Modular automation consists of consolidated analyzers, integrated analyzers, modular workcells, and pre- and post-analytical automation. These terms will be defined in this paper. Using a modular automation model, the automated core laboratory will become a site where laboratory data is evaluated by trained professionals to provide diagnostic information to practising physicians. Modem software information management and process control tools will complement modular hardware. Proper standardization that will allow vendor-independent modular configurations will assure success of this revolutionary new technology.
Automation↗
Renal dopamine receptors in health and hypertension.
During the past decade, it has become evident that dopamine plays an important role in the regulation of renal function and blood pressure. Dopamine exerts its actions via a class of cell-surface receptors coupled to G-proteins that belong to the rhodopsin family. Dopamine receptors have been classified into two families based on pharmacologic and molecular cloning studies. In mammals, two D1-like receptors that have been cloned, the D1 and D5 receptors (known as D1A and D1B, respectively, in rodents), are linked to stimulation of adenylyl cyclase. Three D2-like receptors that have been cloned (D2, D3, and D4) are linked to inhibition of adenylyl cyclase and Ca2+ channels and stimulation of K+ channels. All the mammalian dopamine receptors, initially cloned from the brain, have been found to be expressed outside the central nervous system, in such sites as the adrenal gland, blood vessels, carotid body, intestines, heart, parathyroid gland, and the kidney and urinary tract. Dopamine receptor subtypes are differentially expressed along the nephron, where they regulate renal hemodynamics and electrolyte and water transport, as well as renin secretion. The ability of renal proximal tubules to produce dopamine and the presence of receptors in these tubules suggest that dopamine can act in an autocrine or paracrine fashion; this action becomes most evident during extracellular fluid volume expansion. This renal autocrine/paracrine function is lost in essential hypertension and in some animal models of genetic hypertension; disruption of the D1 or D3 receptor produces hypertension in mice. In humans with essential hypertension, renal dopamine production in response to sodium loading is often impaired and may contribute to the hypertension. The molecular basis for the dopaminergic dysfunction in hypertension is not known, but may involve an abnormal post-translational modification of the dopamine receptor.
Animals↗