WITHDRAWN: RhoB-dependent modulation of early endocytic traffic in Madin-Darby canine kidney cells.
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Journal Article↗
Biomedical subjects
Kenneth W Dunn
Publications and source records attributed to Kenneth W Dunn.
Chronic alcohol drinking alters neuronal dendritic spines in the brain reward center nucleus accumbens.
Alcohol is known to affect glutamate transmission. However, how chronic alcohol affects the synaptic structure mediating glutamate transmission is unknown. Repeated alcohol exposure in a subject with familial alcoholic history often leads to alcohol addiction. The current study adopts alcohol-preferring rats, which are known to develop high drinking. Two-photon microscopy analysis indicates that chronic alcohol of 14 weeks either, under continuous alcohol (C-Alc) or with repeated deprivation (RD-Alc), causes dysmorphology--thickened, beaded, and disoriented dendrites that are reminiscent of reactive astrocytes--in a subpopulation of medium spiny neurons. The density of dendritic spines was found differentially lower in the nucleus accumbens of RD-Alc and C-Alc groups as compared with those of Water groups. Large-sized spines and multiple-headed spines were increased in the RD-Alc group. The NMDA receptor subunit NR1 proteins, as analyzed with Western blot, were upregulated in C-Alc, but not in RD-Alc. The upregulated NMDA receptor subunits of NR1 however, are predominantly a splice variant isoform with truncated exon 21, which is required for membrane-bound trafficking or anchoring into a spine synaptic site. These maladaptations may contribute to the transformation of spines. The changes, in density and head-size of spines and the corresponding NMDA receptors, demonstrated an alteration of microcircuitry for glutamate reception. The current study demonstrates for the first time that chronic alcohol exposure causes structural alteration of dendrites and their spines in the key reward brain region in animals that have a genetic background leading to alcohol addiction.
Alcohol-Induced Disorders, Nervous System↗
Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells.
Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.
Animals↗
Principles of multiphoton microscopy.
Multiphoton fluorescence microscopy is a powerful, important tool in biomedical research that offers low photon toxicity and higher spatial and temporal resolution than other in vivo imaging modalities. The capability to collect images hundreds of micrometers into biological tissues provides an invaluable tool for studying cellular and subcellular processes in the context of tissues and organs in living animals. Multiphoton microscopy is based upon two-photon excitation of fluorescence that occurs only in a sub-femtoliter volume at the focus; by scanning the focus through a sample, 2- and 3-dimensional images can be collected. The complex 3-dimensional organization of the kidney makes it especially appropriate for multiphoton microscopic analysis, which has been used to characterize numerous aspects of renal physiology and pathophysiology in living rats and mice. However, the ability to collect fluorescence images deep into biological tissues raises unique problems not encountered in other forms of optical microscopy, including issues of probe access, and tissue optics. Future improvements in multiphoton fluorescence microscopy will involve optimizing objectives for the unique characteristics of multiphoton fluorescence imaging, improving the speed at which images may be collected and extending the depth to which imaging may be conducted.
Animals↗
Burn injuries inflicted on children or the elderly: a framework for clinical and forensic assessment.
LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Understand the difference between battery and assault in U.S. law and the concepts of the phrase "child abuse" and "elder abuse." 2. Understand that state statutes vary and can define abuse narrowly or with great specificity, and that either definition has inherent problems for physicians treating victims of abuse and neglect. 3. Know where to find the state-specific legal criteria for child or elder abuse and neglect, along with the corresponding standards for mandatory reporting and physician accountability. 4. Understand the relevant law regarding physician-patient privilege and the repercussions of incorrect but good faith reporting and of failing to report suspected abuse or neglect of children or the elderly. 5. Understand that there are no pathognomic signs for inflicted burn injury. 6. Clinically assess burned pediatric or elderly patients within a framework that will minimize the risk of missing or inappropriately suspecting injuries that stem from abuse or neglect. SUMMARY: This article deals with burns inflicted on children and the elderly, two particularly vulnerable societal groups. Though inflicted burning is a relatively rare method of inflicting physical abuse, failure to diagnose it has far-reaching ramifications. These injuries pose both medical and forensic problems for physicians, along with unique ethical dilemmas. This article is a collaboration between surgeons and lawyers providing a holistic, workable approach to the management of inflicted burn injury. The authors first describe the legal considerations that must be appreciated by U.S. physicians, then they suggest a rational and balanced clinical approach to the assessment of burn injuries that may have been inflicted intentionally or negligently on children and the elderly.
Age Factors↗
Estimation of breast burn size.
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Body Mass Index↗
Intentional burn injury: an evidence-based, clinical and forensic review.
Burn injury can be inflicted intentionally either by one person to another whenever one has the ability to physically control the other, or it can be self-inflicted. There is scant evidential basis for much that is written about and practiced in the evaluation and care of patients that have sustained intentional burn injuries. Yet this is an area in which medical personnel must necessarily be trained in both the therapeutic and forensic aspects of a complex problem. Failure to appreciate the complexity of medical and forensic interactions may have far reaching effects. A missed diagnosis can result in inappropriate medical care, on-going abuse and future fatality. Inept management can result on the one hand, in blame levelled inappropriately placing incomparable strain on family units and innocent parties, and on the other, allow abusers to continue unchecked. This is the first review on the subject in which lawyers and doctors collaborate to produce a holistic approach to this subject. In it we describe the legal considerations that medical staff must appreciate when approaching patients who may have suffered intentional burns. We analyse the various scenarios in which intentional burning can be found and challenge the clinical dogma with much of the management of paediatric inflicted burns has become imbued. We suggest a rational and balanced approach to all intentional burn injuries-especially when children are involved. In the light of current case law in which dogmatic medical evidence has been implicated in wrongful convictions for child abuse in the UK, it is imperative that medical professionals gather evidence carefully and completely and apply it with logic and impartiality. This paper will aid clinicians who may not be experienced in dealing with burn injuries, but find themselves in the position of seeing a burn acutely, to avoid common mistakes.
Adult↗
Renal cysts of inv/inv mice resemble early infantile nephronophthisis.
Cystic kidney disease has been linked to mutations in the Invs gene in mice with inversion of embryonic turning (inv/inv) and the INVS (NPHP2) gene in infants with nephronophthisis type 2 (NPHP2). The inv mouse model features multiorgan defects including renal cysts, altered left-right laterality, and hepatobiliary duct malformations transmitted in an autosomal recessive manner. Affected mice usually die of renal and liver failure by postnatal day 7. Although cardiopulmonary and liver anomalies have been carefully detailed, renal cysts have yet to be fully characterized in inv/inv. By use of three-dimensional visualization by two-photon microscopy, this study provides the first comprehensive analysis of in situ cyst formation and progression in inv/inv kidneys. At embryonic day 15, there is dilatation of Bowman's capsule followed temporally by corticomedullary cysts involving collecting ducts, proximal tubules, and thick ascending limbs. Collecting ducts of newborn inv/inv mice are uniformly and diffusely cystic from medulla to cortex, with normal diameters found only at their most proximal tips. Proximal tubules form fusiform cysts that alternate with segments of normal or narrowed caliber along torturous convolutions. Because defective cilia have been linked to situs inversus and cystogenesis, we examined inv/inv cilia by scanning and transmission electron microscopy. The former detected monocilia of expected length in cystic collecting ducts and proximal tubules; the latter demonstrated the usual 9 + 2 pattern in respiratory cilia. The inv mutant mouse has renal cysts resembling infantile NPHP2 and will provide broader insight into the role cilia play in renal cystogenesis.
Animals↗
Two-photon in vivo microscopy of sulfonefluorescein secretion in normal and cystic rat kidneys.
Sulfonefluorescein (SF) is a fluorescent organic anion secreted by kidney proximal tubules. The purposes of this study were 1) to quantify accumulation of SF in normal and cystic rat kidneys in vivo and 2) to test whether SF accumulation could be used as a marker for cysts derived from proximal tubules. Male Munich-Wistar rats, normal Han:SPRD rats, and heterozygous Han:SPRD rats with autosomal-dominant polycystic kidney disease were anesthetized with Inactin and solutions containing SF were administered by constant intravenous infusion. In Munich-Wistar rats, SF fluorescence in the urinary space of Bowman's capsule averaged 0.15 +/- 0.04 (n = 17) times that of glomerular capillary plasma, consistent with extensive plasma protein binding of SF. In normal Han:SPRD rats, steady-state cell cytoplasm SF fluorescence in proximal tubule and distal tubule cells averaged, respectively, 2.7 +/- 1.4 (n = 99 tubules) and 0.2 +/- 0.2 (n = 17) times that of peritubular capillary plasma. No punctate SF fluorescence was seen in proximal tubule cell cytoplasm. Probenecid reduced proximal tubule cell SF fluorescence to 0.64 +/- 0.40 (n = 64) times that of plasma. Ureteral obstruction decreased the proximal tubule cell-to-lumen SF fluorescence gradient, suggesting that tubule fluid flow normally sweeps away secreted SF. In cystic kidneys, cysts derived from proximal tubules could be identified by their uptake of SF, but cell uptake was patchy. We conclude that in vivo two-photon microscopy is a powerful tool for quantifying glomerular and tubular handling of SF, and SF can be used to identify proximal tubule-derived cysts.
Animals↗
The E5 protein of human papillomavirus type 16 perturbs MHC class II antigen maturation in human foreskin keratinocytes treated with interferon-gamma.
Major histocompatibility complex (MHC) class II antigens are expressed on human foreskin keratinocytes (HFKs) following exposure to interferon gamma. The expression of MHC class II proteins on the cell surface may allow keratinocytes to function as antigen-presenting cells and induce a subsequent immune response to virus infection. Invariant chain (Ii) is a chaperone protein which plays an important role in the maturation of MHC class II molecules. The sequential degradation of Ii within acidic endocytic compartments is a key process required for the successful loading of antigenic peptide onto MHC class II molecules. Since human papillomavirus (HPV) 16 E5 can inhibit the acidification of late endosomes in HFKs, the E5 protein may be able to affect proper peptide loading onto the MHC class II molecule. To test this hypothesis, HFKs were infected with either control virus or a recombinant virus expressing HPV16 E5 and the infected cells were subsequently treated with interferon-gamma. ELISAs revealed a decrease of MHC class II expression on the surface of E5-expressing cells compared with control virus-infected cells after interferon treatment. Western blot analysis showed that, in cells treated with interferon gamma, E5 could prevent the breakdown of Ii and block the formation of peptide-loaded, SDS-stable mature MHC class II dimers, correlating with diminished surface MHC class II expression. These data suggest that HPV16 E5 may be able to decrease immune recognition of infected keratinocytes via disruption of MHC class II protein function.
Antigens, Differentiation, B-Lymphocyte↗
Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts.
The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca(2+)](i) transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca(2+)](i) transients were recorded at depths </=100 microm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca(2+)](i) transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t(90-50%)) and from 50 to 10% (t(50-10%)) of the peak [Ca(2+)](i) were (means +/- SE) 73 +/- 4 and 126 +/- 10 ms, respectively, and at 2 Hz, 62 +/- 3 and 94 +/- 6 ms (n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [Ca(2+)](i) decay was markedly slower in fura-2-loaded hearts (t(90-50%) at 1 Hz, 128 +/- 9 ms and at 2 Hz, 88 +/- 5 ms; t(50-10%) at 1 Hz, 214 +/- 18 ms and at 2 Hz, 163 +/- 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [Ca(2+)](i) decline resulted from increased cytosolic Ca(2+) buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca(2+) chelator BAPTA. Propagating calcium waves and [Ca(2+)](i) amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging.
Animals↗
Testing effects of signal transduction pathways on cadherin junctional complex assembly using quantitative image analysis.
Cadherin adhesion molecules function in numerous cell biological processes that influence embryo development, normal cell physiology, and pathophysiology of many disease processes. Cadherins nucleate the assembly of the adherens junction, a cell-to-cell adhesion plaque that is prominent in simple epithelial cells and found in many cell types. Numerous cell biological approaches have been used to study this interesting class of molecules. Here, we outline methodology used in our studies of junctional complexes to examine effects of signaling molecules on assembly mechanisms. This is a quantitative method that allows the investigator to test the combined effect of two different signaling processes to determine whether these two signals act in concert within the same pathway. We discuss how this method could be generalized to other studies to examine consequences of various experimental manipulations on the assembly of cellular structures.
Cadherins↗
Novel light microscopy imaging techniques in nephrology.
PURPOSE OF REVIEW: As more genomes are sequenced, the difficult task of characterizing the gene products of these genomes becomes the compelling mission of biological sciences. The melding of whole organ physiology with transgenic animal models, gene transfer methods and RNA silencing promises to form the next wave of scientific inquiry. A host of new microscopy imaging technologies enables researchers to directly visualize gene products, probe alterations in cell function in transgenic animals and map tissue organization. This review will describe these microscopy imaging techniques, their advantages, imaging properties and limitations. RECENT FINDINGS: New optical methods such as two-photon confocal microscopy, fluorescence resonance energy transfer, and total internal fluorescence reflectance microscopy are increasingly being applied to extend our understanding of whole organ and renal epithelial function. Two-photon confocal microscopy has been used to image directly into the kidney of living animals. Fluorescence resonance energy transfer has been used to directly visualize transcription factor complexes within the nucleus while total internal fluorescence reflectance microscopy has permitted direct observation of protein delivery to the plasma membrane. SUMMARY: The application of these optical techniques along with the ability to label virtually any protein with a fluorescent tag will enable researchers to study cellular processes and whole organ function in vivo. Light microscopy methods will allow an advance from semi-quantitative to quantitative approaches to problems of relevance to physiologists studying issues related to renal function.
Animals↗
Intravital imaging of the kidney using multiparameter multiphoton microscopy.
Intravital optical microscopy provides a powerful means of studying the cell biology in the most physiologically relevant setting. The ability of multiphoton microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of multiple organs. Presented here are examples of how two-photon microscopy can be applied to intravital studies of kidney physiology and the study of disease processes. These include studies of cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters. Two-photon microscopy can now provide a level of investigation previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution. Therefore, application of this technology will provide direct visualization of organ-specific and cell-specific responses to an array of stimuli and therapeutic approaches, enhancing our understanding and treatment of disease processes.
Animals↗
A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis.
Apoptosis is an important mode of cell death under both physiological and pathophysiological conditions. Numerous techniques are available for the study and quantitation of apoptosis in cell culture, but only few are useful when applied to complex tissues. Among these, the terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay remains the most widely used technique. However, its specificity and sensitivity for the detection of apoptosis remain controversial. We developed a technique consisting of staining live cells and tissues with Hoechst 33342 and the vital dye propidium iodide (PI), followed by fixation and the TUNEL reaction. We demonstrate excellent retention of PI in necrotic cells after fixation. We also examined the distribution of TUNEL staining among necrotic and apoptotic cells in various models of cell injury in vitro and in vivo. We show that the sensitivity of the TUNEL varied between 61 and 90% in the models examined. The specificity exceeded 87% in all models but fell to 70% when a predominantly necrotic injury was induced. This novel and simple method will permit the determination of indices of sensitivity and specificity for the TUNEL assay in other tissues and experimental conditions.
Animals↗
Nucleotide depletion increases trafficking of gentamicin to the Golgi complex in LLC-PK1 cells.
Having shown rapid trafficking of aminoglycosides to the Golgi complex in cell culture, we focused on the injurious interaction that occurs when gentamicin administration is preceded by renal ischemia. Using Texas red-labeled gentamicin as a tracer, we determined that 15 min of cellular nucleotide depletion did not significantly increase subsequent uptake. However, cells previously depleted of nucleotides accumulated significantly more Texas red-labeled gentamicin within a dispersed Golgi complex. Using Ricinus communis and Lens culinaris lectins, which label specific compartments of the Golgi complex (trans-Golgi network/trans and medial/cis compartments, respectively), we determined that the medial/cis compartment dispersed after 15 min of nucleotide depletion but the trans-Golgi network/trans compartment remained unaffected. An increase in the number of cells exhibiting disrupted medial/cis-Golgi morphology after repletion in physiological media containing gentamicin was also seen. In summary, the increase in nephrotoxicity seen when ischemia precedes aminoglycoside uptake may be part of a complex mechanism initially involving increased Golgi accumulation and prolonged Golgi dispersion. The Golgi complex must then endure the effects of gentamicin accumulated in larger quantities in an aberrant physiological state.
Animals↗
Functional studies of the kidney of living animals using multicolor two-photon microscopy.
Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.
Animals↗
Rac1, but not RhoA, signaling protects epithelial adherens junction assembly during ATP depletion.
Rho family GTPase signaling regulates actin cytoskeleton and junctional complex assembly. Our previous work showed that RhoA signaling protects tight junctions from damage during ATP depletion. Here, we examined whether RhoA GTPase signaling protects adherens junction assembly during ATP depletion. Despite specific RhoA signaling- and ATP depletion-induced effects on adherens junction assembly, RhoA signaling did not alter adherens junction disassembly rates during ATP depletion. This shows that RhoA signaling specifically protects tight junctions from damage during ATP depletion. Rac1 GTPase signaling also regulates adherens junction assembly and therefore may regulate adherens junction assembly during ATP depletion. Indeed, we found that Rac1 signaling protects adherens junctions from damage during ATP depletion. Adherens junctions are regulated by various GTPases, including RhoA and Rac1, but adherens junctions are specifically protected by Rac1 signaling.
Adenosine Triphosphate↗