MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This article explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicine and cytochalasin B, on the degree of tightness of the occluding junctions . To study the degree of tightness, monolayers were prepared by plating MDCK cells on nylon disks coated with collagen . Disks were mounted as flat sheets between two Lucite chambers, and the'sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers . Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation . Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane . Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell . Colchicine causes a profound disruption of microtubules but only a 27% decrease in the electrical resistance of the resting monolayers . Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance . The cortical ring of filaments remains in place but appears fragmented with time . We find that removal of extracellular Ca", which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca". Colchicine has no effect on either the opening or reversal processes, but cytochalasin B inhibits the resealing of the junctions by disorganizing the filaments in the ring and at the apical border of the cells. These cytochalasin B effects are fully reversible . The correlation among cell shape, cytoskeletal patterns, and electrical resistance in the EGTAopened and resealed monolayers suggests that microfilaments, through their association with plasma membrane components, play a role in positioning the junctional strands and influence the degree of sealing of the occluding junctions.

In MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring. We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB. In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB-treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In additions we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with Mr of greater than or equal to 120K daltons as well as peptides with Mr = 56K, 50K, 36K, and 18K daltons.

Swelling of cultured astrocytes exposed to hyposmotic medium modified the organization of the filamentous actin (F-actin) cytoskeleton, making the actin network diffuse in the cell body but concentrated at foci corresponding to the tips of the cell projections retracted by swelling. This change was reversible, and, after 2 h, the actin cytoskeleton tended to recover, and cells regained their flat and stellate shape. Cytochalasins B and D (CB and CD, respectively), which disrupt the actin cytoskeleton, did not affect regulatory volume decrease (RVD) or the swelling-activated efflux of Cl- and inositol, although 10 microM CD increased the basal efflux of taurine. The mercurial p-chloromercuribenzenesulfonate (0.5-1 mM), known to disrupt the membrane cytoskeleton in isosmotic conditions, induced a 46, 50, and 38% release of [3H]taurine, 125I, and [3H]inositol, respectively, causing cell shrinkage and retraction of the cytoskeleton. Coincidently, the swelling-stimulated release of [3H]taurine and 125I was reduced by 60 and 30%, respectively. Results of this study do not exclude the possibility that changes in the actin cytoskeleton elicited by swelling are involved in mechanisms of RVD and only indicate that the disruption caused by cytochalasins is unrelated to that process.

Alterations in birth weight impact postnatal outcome and adult metabolic health. Therefore, fetal growth regulation is crucial for preventing chronic metabolic diseases. Leptin has been suggested to play an important role in placental and fetal growth, albeit its specific mechanisms of action have not been elucidated. The aim of this study was to analyze leptin concentrations in placenta, cord blood, and maternal blood of SGA, AGA, and LGA (small, adequate and large for gestational age, respectively) newborns, as well as placental leptin receptor (LEPRa and LEPRb) protein expression. We performed a cross-sectional comparative study in 3 groups of healthy mothers and their term newborns at delivery (SGA, AGA, and LGA, n=20 per group). Placental, maternal blood, and cord blood leptin content were measured by ELISA. Placental LEPRa and LEPRb protein expression were determined by Western Blot. Maternal leptin concentrations correlated positively with maternal weight before and at the end of gestation, without differences between groups. Cord leptin is higher in LGA and lower in SGA, whereas placental leptin is higher in SGA. Placental leptin was inversely correlated with placental weight, independently from maternal weight and gestational age. Both LEPRa and LEPRb expression are lower in SGA, while LEPRa positively correlated with placental weight and birthweight. The current findings indicate that placental leptin and its receptors are differentially expressed in SGA, AGA, and LGA newborns. We suggest that placental leptin and LEPR protein expression may influence placental growth and thus, birth weight.

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis. This parasite invades its host by penetrating the olfactory mucosa. However, the mechanism of epithelium penetration is not well understood. In the present study, we evaluated the effect of N. fowleri trophozoites and the non-pathogenic Naegleria gruberi on Madin-Darby canine kidney (MDCK) tight junction proteins, including claudin-1, occludin and ZO-1, as well as on the actin cytoskeleton. Trophozoites from each of the free-living amoeba species were co-cultured with MDCK cells in a 1 : 1 ratio for 1, 3, 6 or 10 h. Light microscopy revealed that N. fowleri caused morphological changes as early as 3 h post-infection in an epithelial MDCK monolayer. Confocal microscopy analysis revealed that after 10 h of co-culture, N. fowleri trophozoites induced epithelial cell damage, which was characterized by changes in the actin apical ring and disruption of the ZO-1 and claudin-1 proteins but not occludin. Western blot assays revealed gradual degradation of ZO-1 and claudin-1 as early as 3 h post-infection. Likewise, there was a drop in transepithelial electrical resistance that resulted in increased epithelial permeability and facilitated the invasion of N. fowleri trophozoites by a paracellular route. In contrast, N. gruberi did not induce alterations in MDCK cells even at 10 h post-infection. Based on these results, we suggest that N. fowleri trophozoites disrupt epithelial monolayers, which could enable their penetration of the olfactory epithelium and subsequent invasion of the central nervous system.