Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy

Summary MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450–500 nm excitation/>510 nm emission) or rhodamine (514 nm excitation/>530 emission) type dyes. A second 5- to 10-fold brighter emission for 450–500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled domes and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 m riboflavin resulted in accumulation in domes of 565±80 m. The transport rate was calculated to be 189±30 pmol/min-cm². Onemm probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10mm nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatable with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.     

Detection of cytomegalovirus in urine of HIV-infected patients by DNA-DNA hybridization comparison with virus isolation, immunofluorescence and immunoperoxidase.

Immunofluorescence and immunoperoxidase test directed against early viral antigens, and DNA-DNA hybridization were compared with viral isolation for their abilities to detect Cytomegalovirus (CMV) in the urine of 89 HIV infected patients. From the 100 urine samples collected, 70 were found positive by at least one method. Considering viral isolation as the "gold standard" technique, immunofluorescence and immunoperoxidase had a sensitivity of 92.3% and 88% respectively, with a specificity in both cases of 95%. DNA-DNA hybridization showed a sensitivity of 90% but with lower (60%) specificity. All of the three assays were effective in detecting CMV from urine and the technical advantage of each is discussed.     

Transfer of a plasmid determining bacteriocin Bc-48 production and immunity, and response to sexual pheromones in Enterococcus faecalis S-48.

Production of bacteriocin Bc-48 by Enterococcus faecalis S-48 is encoded by the conjugative plasmid pMB1, which is approximately 90 kb and also responds to sex pheromones of E. faecalis OG1X. Mutants harboring deleted forms of this plasmid (pMB1-del, 75 kb) have lost both the phenotype Bc-48 (production and immunity) and the clumping response. The conjugal transfer of pMB1 to E. faecalis OG1X results in the acquisition by this strain of both bacteriocin production and immunity and also the clumping response. In the transconjugants isolated, the bacteriocinogenic trait is associated with a smaller plasmid (52 kb), which we call pMB1-1. The relationship among plasmids pMB1, pMB1-del, and pMB1-1 has been demonstrated by DNA hybridization. Plasmid pMB1-1 has been transferred with high frequency to E. faecalis mutants cured of Bc-48 production (carrying pMB1-del), conferring to them the Bc-48 trait and clumping response. In the transconjugants from a second mating, pMB1-1 and pMB1-del coexist without appreciable segregation.     

Up-regulated RxLR effector genes of Plasmopara viticola in synchronized host-free stages and infected leaves of hosts with different susceptibility

Fast recognition of host signals and early activation of infection mechanisms in Plasmopara viticola are decisive for successful infestation of Vitis vinifera. To better understand interactive processes at the first front line of combat between the pathogen and its host, a specific pre-infective stage was generated in a host-free system. Zoospore encystment was triggered within minutes after treatment with CaCl₂. Subsequently, high rates of germ tube formation occurred in a synchronized manner. This method was employed to compare development-related gene expression in strains of different virulence. Soon after germination, spores showed strong up-regulation of two effector genes, PvRxLR18 and PvRxLR28, particularly in the high virulence strain. On infected grapevine leaf-discs of cultivars with different susceptibility, a similar up-regulation was found at 6 hours post inoculation (hpi). This effect was much more evident in the high virulence than in the low virulence strain and was significantly higher on leaves of the tolerant cultivar Regent than on Müller-Thurgau. In addition, PvRxLR67 was up-regulated 24 hpi in the high virulence strain indicating that different effectors are active in later infection stages. Differences in the expression pattern of RxLR effector genes between the two strains corroborated with infection symptoms visible by sporulation.     

Novel non-ATP competitive small molecules targeting the CK2 α/β interface

Increased CK2 levels are prevalent in many cancers. Combined with the critical role CK2 plays in many cell-signaling pathways, this makes it a prime target for down regulation to fight tumour growth. Herein, we report a fragment-based approach to inhibiting the interaction between CK2α and CK2β at the α-β interface of the holoenzyme. A fragment, CAM187, with an IC₅₀ of 44?μM and a molecular weight of only 257?gmol^?1 has been identified as the most promising compound. Importantly, the lead fragment only bound at the interface and was not observed in the ATP binding site of the protein when co-crystallised with CK2α. The fragment-like molecules discovered in this study represent unique scaffolds to CK2 inhibition and leave room for further optimisation.     

Hydraulic Properties of MDCK Cell Epithelium

The water permeability of the apical and basolateral cell membranes and the compliance of the lateral intercellular spaces (LIS) of MDCK monolayers were measured on confluent cultures grown on permeable supports. Cell membrane water permeabilities were determined, using quantitative differential interference light microscopy, from the rate of cell volume decrease after exposure to a hyperosmotic bathing solution. Both membranes exhibited osmotic water permeabilities (P_OSM) of ∼10 μm/sec, comparable to that of unmodified lipid bilayers. The compliance of the cell membranes forming the lateral intercellular space (LIS) between cells was determined from the pressure-volume relation. Confocal microscopy of fluorescent labeling of the basolateral cell membranes was used to delineate the LIS geometry as transepithelial hydrostatic pressure was varied. The LIS were poorly deformable as a function of transepithelial hydrostatic pressure until a pressure of ≥8 cm H₂O (basolateral > apical) was reached where catastrophic failure of intercellular connections occurred. The compliance of the LIS was calculated from the geometry changes at pressures <8 cm H₂O and ranged from 0.05–0.11 cm H₂O⁻¹, comparable to that previously predicted in mathematical models of the rat proximal tubule. Received: 10 January 1996/Revised: 9 May 1996     

The chloride concentration in the lateral intercellular spaces of MDCK cell monolayers

We measured the Cl concentration of the lateral intercellular spaces (LIS) of MDCK cell monolayers, grown on glass coverslips, by video fluorescence microscopy. Monolayers were perfused at 37°C either with HEPES-buffered solutions containing 137 mm Cl or bicarbonate/CO₂-buffered solutions containing 127 mm Cl. A mixture of two fluorescent dyes conjugated to dextrans (MW 10,000) was microinjected into domes and allowed to diffuse into the nearby LIS. The Cl sensitive dye, ABQ-dextran, was selected because of its responsiveness at high Cl concentrations; a Clinsensitive dye, Cl-NERF-dextran, was used as a reference. Both dyes were excited at 325 nm, and ratios of the fluorescence intensity at spectrally distinct emission wavelengths were obtained from two intensified CCD cameras, one for ABQ-dextran the other for Cl-NERFdextran. LIS Cl concentration was calibrated in situ by treating the monolayer with digitonin or ouabain and varying the perfusate Cl between 0 and 137 mm (HEPES buffer) or between 0 and 127 mm (bicarbonate/CO₂ buffer). LIS Cl in HEPES-buffered solutions averaged 176 ± 19 mm (n = 12), calibrated with digitonin, and 170 ± 9 mm (n = 12), calibrated with ouabain. LIS Cl in bicarbonate/CO₂-buffered solutions averaged 174 ± 10 mm (n = 7) using the ouabain calibration. The Cl concentration of MDCK cell domes, measured with Clsensitive microelectrodes and by microspectrofluorimetry, did not differ significantly. Images of the LIS at 3 focal planes, near the tight junction, midway and basal, failed to reveal any gradients in Cl concentration along the LIS. LIS Cl changed rapidly in response to perfusate Cl with characteristic times of 0.8 ± 0.1 min (n = 21) for Cl decrease and 0.3 ± 0.04 min (n=21) for Cl increase. In conclusion, (i) Cl concentration is higher in the LIS than in the bathing medium, (ii) no gradients of Cl along the depth of LIS are detectable, (iii) junctional Cl permeability is high.We gratefully acknowledge the assistance of Mr. Richard D'Alessandro in the performance of the microelectrode studies. Mr. Carter Gibson designed the electronics and wrote the key computer programs used in this study. The authors are grateful to Dr. Alan Verkman (UCSF) for his advice and gifts of fluorescent probes in the early stages of this work.     

Bacterial Production and Purification of the Fish Pituitary Hormone Somatolactin

Somatolactin, a pituitary hormone belonging to the growth hormone/prolactin family, is produced in the intermediate lobe of teleost pituitary. To date, the functions of this new hormone and the target tissues are unknown. ASolea senegalensissomatolactin (ssSL) cDNA has previously been cloned and isolated. Here we have inserted this cDNA into a pET-3a plasmid in order to produce recombinant ssSL inE. coliBL21 (DE3) cells. The protein induced was isolated from inclusion bodies by a solubilization–renaturation procedure originally developed to generate native disulfide bonds, to get putative active proteins. The recombinant somatolactin was further purified to homogeneity by gel filtration on FPLC. The estimated molecular weight of 26 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis agrees well with the molecular mass calculated from the translated cDNA sequence and with native somatolactin (SL). The recombinant protein showed electrophoretic mobility identical to that of one of the native forms of SL secretedin vitroby cultured pituitaries from sole. Another native SL expressed inS. senegalensisrepresented a glycosylated modified hormone as shown byN-glycosidase treatment. Further, recombinant SL was recognized by an anti-native SL antibody and used to generate polyclonal sera reactive with the native pituitary hormone. To date, this represents the first recombinant SL protein isolated in sufficient quantities for biophysical and biochemical investigation and for studies on its physiological actions.