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.     

Karyotypic study of titi monkeys,Callicebus moloch brunneus

A karyotypic study on a subspecies of the dusky titi,Callicebus moloch brunneus, was carried out and a third karyotype ofC. moloch was discovered. The chromosome number of this subspecies is 48. The autosomes consist of 5 subtelocentric, 5 submeta- or metacentric, and 13 acrocentric chromosome pairs. The X chromosome and the Y chromosome are submetacentric and metacentric, respectively. A comparative study with other subspecies of theC. moloch group (i.e.,C. m. cupreus andC. m. ornatus with 2n=46 andC. m. donacophilus with 2n=50) suggests that the karyotype ofbrunneus occupies a position intermediate between the two other karyotypes ofC. moloch, but nearer to that of 2n=50. The presumed total differences betweenbrunneus andcupreus comprise one Robertsonian rearrangement, one centromeric transposition and four pericentric inversions, and those betweenbrunneus anddonacophilus involve one translocation or breakage (possibly corresponding to two events, that is, one Robertsonian rearrangement and one centromeric transposition).     

Morphology of the nucleo-cytoplasmic interactions during the development ofAcetabularia cells

Protoplasma - An electron microscopic survey of nuclear events and changes in the perinuclear cytoplasm during the generative phase ofAcetabularia is presented with details on late stages in the...     

A parallel path model forNecturus proximal tubule

Summary A parallel path model based on the principles of nonequilibrium thermodynamics was developed for theNecturus proximal tubule. The cellular path was represented as a luminal membrane followed by an irreversible active NaCl transport system in the peritubular barrier. The shunt pathway was described as three coarse barriers in series: tight junction, lateral intercellular spaces, and basement membrane with connective tissue. Volume and solute flows were predicted by the model equations as a function of applied electric current. Variations of the model parameters revealed the quantitative importance of the shunt path properties and the relative insensitivity of epithelial transport to changes in most cell parameters. Circulation of electric current and solute within the epithelium were shown to significantly influence the bahavior of the tubule in the presence of an electric field. Values for all transport parameters of the shunt path and epithelium were calculated and compared with available experimental evidence. Volume flow and electric currents predicted by the model compared favorably with experimental observations.     

Herpes Simplex Virus Products in Productive and Abortive Infection I. Stabilization with Formaldehyde and Preliminary Analyses by Isopycnic Centrifugation in CsCl

Lysates of HEp-2 cells productively infected with herpes simplex virus yielded two bands on isopycnic centrifugation in CsCl gradients, ranging from 1.2 to 1.6 g/cm(3). One band, designated alpha, had a mean buoyant density of 1.27 g/cm(3) and contained herpes virions. Band beta had a mean density of 1.305 g/cm(3) and contained primarily complement-fixing viral antigens and little or no viral deoxyribonucleic acid (DNA). The products banding in the alpha and beta bands were unstable; fivefold or higher amounts were recovered by treating the cell extract with formaldehyde prior to centrifugation. Formaldehyde treatment increased the buoyant density of viral products in both the alpha and beta bands by about 0.015 g/cm(3). In addition, it stabilized hitherto inapparent products, forming a broad band gamma with a density range of 1.37 to 1.45 g/cm(3). The material in the gamma band was heterogeneous; it contained viral DNA, cellular DNA, and viral antigen. Formalinized lysates of DK cells abortively infected with herpes simplex virus yielded a beta band undifferentiated from that formed by extracts of productively infected cells. The gamma band was less dense and narrower. The alpha band was entirely missing.     

Compound kinetochores of the Indian muntjac

The chromosomes of the Indian muntjac (Muntiacus muntjak vaginalis) are unique among mammals due to their low diploid number (2N=6, 7) and large size. It has been proposed that the karyotype of this small Asiatic deer evolved from a related deer the Chinese muntjac (Muntiacus reevesi) with a diploid chromosome number of 2n= 46 consisting of small telocentric chromosomes. In this study we utilized a kinetochore-specific antiserum derived from human patients with the autoimmune disease scleroderma CREST as an immunofluorescent probe to examine kinetochores of the two muntjac species. Since CREST antiserum binds to kinetochores of mitotic chromosomes as well as prekinetochores in interphase nuclei, it was possible to identify and compare kinetochore morphology throughout the cell cycle. Our observations indicated that the kinetochores of the Indian muntjac are composed of a linear beadlike array of smaller subunits that become revealed during interphase. The kinetochores of the Chinese muntjac consisted of minute fluorescent dots located at the tips of the 46 telocentric chromosomes. During interphase, however, the kinetochores of the Chinese muntjac clustered into small aggregates reminiscent of the beadlike arrays seen in the Indian muntjac. Morphometric measurements of fluorescence indicated an equivalent amount of stained material in the two species. Our observations indicate that the kinetochores of the Indian muntjac are compound structures composed of linear arrays of smaller units the size of the individual kinetochores seen on metaphase chromosomes of the Chinese muntjac. Our study supports the notion that the kinetochores of the Indian muntjac evolved by linear fusion of unit kinetochores of the Chinese muntjac. Moreover, it is concluded that the evolution of compound kinetochores may have been facilitated by the nonrandom aggregation of interphase kinetochores in the nuclei of the ancestral species.