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Robert L Bacallao

Publications and source records attributed to Robert L Bacallao.

10 recordsLinked to original sources
PubMed2006

Imaging gene expression.

Genome-wide sequencing and increasing use of microarrays has resulted in the identification of a large number of new genes which may have important functional roles in development and onset of disease. Classical approaches in gene expression studies fall short in providing information about cellular localization of these genes and their relative levels of expression in tissues. Rapid determination of gene expression can be achieved with in situ methods of localization of gene expression in combination with recent developments in imaging technology.

Animals↗
PubMed2006

Pulmonary immunity to viral infection: adenovirus infection of lung dendritic cells renders T cells nonresponsive to interleukin-2.

Adenovirus (Ad) infection has been identified as predisposing hosts to the development of pulmonary disease through unknown mechanisms. Lung dendritic cells (DCs) are vital for initiating pulmonary immune responses; however, the effects of Ad infection on primary lung DC have not been studied. In contrast to the effects on bone marrow- and monocyte-derived DCs, the current study shows that Ad infection of murine BALB/c lung DCs in vitro and in vivo suppresses DC-induced T-cell proliferation. The effect of Ad on DCs was not due to a downregulation of major histocompatibility complex or costimulatory molecules. Analysis of the production of interleukin-12 (IL-12), alpha interferon (IFN-alpha), and IFN-gamma by the Ad-infected DCs shows no significant differences over noninfected control lung DCs. Ad-induced suppression was not due to a deficiency of IL-2 or other DC-secreted factors and was dependent on viral protein synthesis, as UV irradiation of Ad abrogated the suppressive effect. Results suggest that Ad-infected DCs induce T cells to be nonresponsive to IL-2 during primary coculture, as the addition of IL-2 in secondary cultures recovered T-cell proliferation. In vivo studies supported in vitro results showing that Ad infection resulted in lung T cells with decreased proliferative ability. This study demonstrates that Ad infection induces local immunoincompetence by altering DC-T-cell interactions.

Adenoviridae Infections↗
PubMed2004

Real-time three-dimensional imaging of lipid signal transduction: apical membrane insertion of epithelial Na(+) channels.

In the distal tubule, Na(+) resorption is mediated by epithelial Na(+) channels (ENaC). Hormones such as aldosterone, vasopressin, and insulin modulate ENaC membrane targeting, assembly, and/or kinetic activity, thereby regulating salt and water homeostasis. Insulin binds to a receptor on the basal membrane to initiate a signal transduction cascade that rapidly results in an increase in apical membrane ENaC. Current models of this signaling pathway envision diffusion of signaling intermediates from the basal to the apical membrane. This necessitates diffusion of several high-molecular-weight signaling elements across a three-dimensional space. Transduction of the insulin signal involves the phosphoinositide pathway, but how and where this lipid-based signaling pathway controls ENaC activity is not known. We used tagged channels, biosensor lipid probes, and intravital imaging to investigate the role of lipids in insulin-stimulated Na(+) flux. Insulin-stimulated delivery of intracellular ENaC to apical membranes was concurrent with plasma membrane-limited changes in lipid composition. Notably, in response to insulin, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) formed in the basolateral membrane, rapidly diffused within the bilayer, and crossed the tight junction to enter the apical membrane. This novel signaling pathway takes advantage of the fact that the lipids of the plasma membrane's inner leaflet are not constrained by the tight junction. Therefore, diffusion of PIP(3) as a signal transduction intermediate occurs within a planar surface, thus facilitating swift responses and confining and controlling the signaling pathway.

Animals↗
PubMed2004

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↗
PubMed2003

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↗
PubMed2002

Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.

Almost 1% of human infants are born with urogenital abnormalities, many of which are linked to irregular connections between the distal ureters and the bladder. During development, ureters migrate by an unknown mechanism from their initial integration site in the Wolffian ducts up to the base of the bladder in a process that we call ureter maturation. Rara(-/-) Rarb2(-/-) mice display impaired vitamin A signaling and develop syndromic urogenital malformations similar to those that occur in humans, including renal hypoplasia, hydronephrosis and mega-ureter, abnormalities also seen in mice with mutations in the proto-oncogene Ret. Here we show that ureter maturation depends on formation of the 'trigonal wedge', a newly identified epithelial outgrowth from the base of the Wolffian ducts, and that the distal ureter abnormalities seen in Rara(-/-) Rarb2(-/-) and Ret(-/-) mutant mice are probably caused by a failure of this process. Our studies indicate that formation of the trigonal wedge may be essential for correct insertion of the distal ureters into the bladder, and that these events are mediated by the vitamin A and Ret signaling pathways.

Animals↗
PubMed2002

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↗
PubMed2002

Inversin forms a complex with catenins and N-cadherin in polarized epithelial cells.

Nephrogenesis starts with the reciprocal induction of two embryonically distinct analages, metanephric mesenchyme and ureteric bud. This complex process requires the refined and coordinated expression of numerous developmental genes, such as inv. Mice that are homozygous for a mutation in the inv gene (inv/inv) develop renal cysts resembling autosomal-recessive polycystic kidney disease. The gene locus containing inv has been proposed to serve as a common modifier for some human and rodent polycystic kidney disease phenotypes. We generated polyclonal antibodies to inversin to study its subcellular distribution, potential binding partners, and functional aspects in cultured murine proximal tubule cells. A 125-kDa inversin protein isoform was found at cell-cell junctions. Two inversin isoforms, 140- and 90-kDa, were identified in the nuclear and perinuclear compartments. Plasma membrane allocation of inversin is dependent upon cell-cell contacts and was redistributed when cell adhesion was disrupted after incubation of the cell monolayer with low-calcium/EGTA medium. We further show that the membrane-associated 125-kDa inversin forms a complex with N-cadherin and the catenins. The 90-kDa nuclear inversin complexes with beta-catenin. These findings indicate that the inv gene product functions in several cellular compartments, including the nucleus and cell-cell adhesion sites.

Animals↗
PubMed2002

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↗