Aucubin supplementation alleviate diabetes induced-disruption of blood-testis barrier and testicular damage via stabilizing cell junction integrity.

Disruption of blood-testis barrier (BTB) was a crucial pathological feature of diabetes induced-testicular injury at early phase. Aucubin (AU), a main active component in Eucommiae Cortex, has drawn attention for its benefits against male reproductive system disease. The current study was aimed at investigating the protective role of AU and exploring the underlying mechanism in diabetic model. A murine model of type 2 diabetes mellitus (T2DM) was induced by high-fat diet (HFD) combined with streptozocin (STZ). Testicular weight index and morphology, sperm quality, integrity of BTB and protein levels were analyzed. The underlying mechanism of the protective effect of AU was further explored in Sertoli cells (SCs) cultured with high glucose (HG). Our results showed AU inhibited testicular structural destruction, restored disruption of BTB and improved abnormal spermatogenic function in diabetic mice. Consistent with in vivo results, HG induced decreased transcellular resistance and increased permeability in SCs monolayers, while AU exposure reverses this trend. Meanwhile, reduced expression of Zonula occludin-1(ZO-1) and Connexin43(Cx43) in testicular tissue diabetic mice and HG-induced SCs was prominently reversed via AU treatment. Mechanistic studies suggested a high affinity interaction between AU and c-Src protein was identified based on molecular docking, and the activation of c-Src was significantly inhibited in AU treatment. Furthermore, AU significantly increased the expression of Cx43 and ZO-1 proteins HG-induced SCs, which can be further enhanced in gene-silenced c-Src cells to some extent. Our results suggested that AU ameliorated disruption of BTB and spermatogenesis dysfunction in diabetic mice via inactivating c-Src to stabilize cell junction integrity.

Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata).

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.

Stabilization of cell-cell junctions by active vitamin D ameliorates uraemia-induced loss of human endothelial barrier function.

Background: Uraemia induces endothelial cell (EC) injury and impaired repair capacity, for which the underlying mechanism remains unclear. Active vitamin D (VD) may promote endothelial repair, however, the mechanism that mediates the effects of VD in chronic kidney disease are poorly understood. Thus, we investigated uraemia-induced endothelial damage and the protection against such damage by active VD. Methods: We applied electric cell-substrate impedance sensing (ECIS) to study real-time responses of human ECs exposed to pooled uraemic and non-uraemic plasma with or without the addition of active VD. The effects of indoxyl sulphate and p-cresol were tested in non-uraemic plasma. Structural changes for vascular endothelial (VE)-cadherin and F-actin were assessed by immunostaining and quantified. Results: The exposure of ECs to uraemic media significantly decreased endothelial barrier function after 24 h. Cell migration after electrical wounding and recovery of the barrier after thrombin-induced loss of integrity were significantly impaired in uraemic-medium stimulated cells and cells exposed to indoxyl sulphate and p-cresol. This effect on ECIS was dependent on loss of cell-cell interaction. Mechanistically, we found that EC, exposed to uraemic media, displayed disrupted VE-cadherin interactions and F-actin reorganization. VD supplementation rescued both endothelial barrier function and cell-cell interactions in ECs exposed to uraemic media. These events were associated with an increment of VE-cadherin at intercellular junctions. Conclusions: Our data demonstrate a potentially clinically relevant mechanism for uraemia-induced endothelial damage. Furthermore, active VD rescued the uraemic medium-induced loss of cell-cell adhesion, revealing a novel role of active VD in preservation of endothelial integrity during uraemia.

Vitamin D Attenuates Endothelial Dysfunction in Uremic Rats and Maintains Human Endothelial Stability.

Background Dysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease ( CKD ). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD , not only by direct effects on the endothelium but also by an increment of α-Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia-induced endothelial damage and the extent to which this was dependent on increased α-Klotho concentrations. Methods and Results In a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial-gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α-Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real-time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro-permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial-cadherin-based cell-cell junctions and diminished F-actin stress fiber organization, preventing the formation of endothelial intracellular gaps. Conclusions Our results demonstrate that paricalcitol attenuates the CKD -induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell-cell interactions.

Role of vitamin D on gut microbiota in cystic fibrosis.

This review explores the potential for vitamin D to favorably alter the gut microbiota, given emerging evidence of the role of vitamin D in controlling mucosal inflammation in the gut. It will focus on cystic fibrosis (CF) patients, a population with both vitamin D deficiency due to gut malabsorption and an altered gut microbiota composition. Recent evidence shows that vitamin D acts to maintain the integrity of the gut mucosal barrier by enhancement of intercellular junctions that control mucosal permeability and reduction of pro-inflammatory cytokines such as IL-8. In addition, vitamin D receptor-mediated signaling has been shown to inhibit inflammation-induced apoptosis of intestinal epithelial cells. As a result of these effects on the intestinal mucosa, maintenance of sufficient vitamin D status may be essential for the development of a healthy gut microbiota, particularly in conditions defined by chronic mucosal inflammation such as CF. We hypothesize here that high dose vitamin D may be used to favorably manipulate the aberrant mucosa seen in patients with CF. This may result in improved clinical outcomes in association with a low inflammatory environment that allows beneficial bacteria to outcompete opportunistic pathogens. Current evidence is sparse but encouraging, and additional evidence is needed to establish vitamin D as a therapeutic approach for gut microbiota modification.

Zinc strengthens the jejunal barrier by reversibly tightening the paracellular route.

During the postweaning period, piglets are prone to gastrointestinal infections. The resulting impairment of intestinal barrier function may cause diarrhea associated with growth retardation or even death of piglets. Orally applied Zn is commonly used to prevent and treat diarrhea, but its mode of action still needs to be elucidated. To analyze the molecular mechanism whereby Zn acts on porcine intestinal barrier function, ex vivo studies on piglet jejunum and accompanying in vitro studies on a porcine jejunal epithelial cell line, IPEC-J2/PS, were performed with electrophysiological tools. Feeding pharmacological Zn doses exerted no significant electrophysiologically ascertainable short- and long-term effects on jejunal barrier function ex vivo. However, in IPEC-J2/PS, basolateral Zn was cytotoxic since its application caused a release of lactate dehydrogenase and an irreversible breakdown of the epithelial barrier. In contrast, apical Zn application caused an immediate increase in paracellular resistance and a decrease in permeability to the paracellular marker fluorescein, reflecting overall barrier strengthening in vitro. Apical effects were fully reversible upon washout. This indicates that Zn supplemented to feed was completely washed out during ex vivo jejunum preparation. We conclude that there is no evidence for long-term barrier effects through prophylactic Zn supplementation and that extracellular Zn acts acutely and reversibly from the apical side via tightening the paracellular route, thus counteracting leak-flux diarrhea.NEW & NOTEWORTHY Therapeutically administered Zn successfully treats diarrhea in veterinary and human medicine. Here we present data that Zn strengthens the porcine jejunal epithelial barrier by reversibly tightening the paracellular route for inorganic ions and small solutes. Acute or long-lasting Zn effects on transcellular transport (Cl- secretion) were not detected. We therefore conclude that Zn is useful for acutely treating leak-flux diarrhea rather than secretory diarrhea. Suitability as prophylactic feed supplement, however, is questionable.

Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates.

The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2 cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2 cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported in vivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by pharmaceutical companies without major changes to their current testing protocols.

Connexin43 hemichannels contributes to the disassembly of cell junctions through modulation of intracellular oxidative status.

Connexin (Cx) hemichannels regulate many cellular processes with little information available regarding their mechanisms. Given that many pathological factors that activate hemichannels also disrupts the integrity of cellular junctions, we speculated a potential participation of hemichannels in the regulation of cell junctions. Here we tested this hypothesis. Exposure of renal tubular epithelial cells to Ca2+-free medium led to disassembly of tight and adherens junctions, as indicated by the reduced level of ZO-1 and cadherin, disorganization of F-actin, and severe drop in transepithelial electric resistance. These changes were preceded by an activation of Cx43 hemichannels, as revealed by extracellular efflux of ATP and intracellular influx of Lucifer Yellow. Inhibition of hemichannels with chemical inhibitors or Cx43 siRNA greatly attenuated the disassembly of cell junctions. Further analysis using fetal fibroblasts derived from Cx43 wide-type (Cx43+/+), heterozygous (Cx43+/-) and knockout (Cx43-/-) littermates showed that Cx43-positive cells (Cx43+/+) exhibited more dramatic changes in cell shape, F-actin, and cadherin in response to Ca2+ depletion, as compared to Cx43-null cells (Cx43-/-). Consistently, these cells had higher level of protein carbonyl modification and phosphorylation, and much stronger activation of P38 and JNK. Hemichannel opening led to extracellular loss of the major antioxidant glutathione (GSH). Supplement of cells with exogenous GSH or inhibition of oxidative sensitive kinases largely prevented the above-mentioned changes. Taken together, our study indicates that Cx43 hemichannels promote the disassembly of cell junctions through regulation of intracellular oxidative status.

Potent In Vitro Protection Against PM2.5-Caused ROS Generation and Vascular Permeability by Long-Term Pretreatment with Ganoderma tsugae.

Epidemiological studies show increased particulate matter (PM[Formula: see text]) particles in ambient air are correlated with increased myocardial infarctions. Given the close association of capillaries and alveoli, the dysfunction is caused when inhaled PM[Formula: see text] particles come in close proximity to capillary endothelial cells. We previously suggested that the inhalation of PM[Formula: see text] diesel exhaust particles (DEP) induces oxidative stress and upregulates the Nrf2/HO-1 pathway, inducing vascular permeability factor VEGFA secretion, which results in cell-cell adherens junction disruption and PM[Formula: see text] transmigratation into circulation. Here, we minimized the level that PM[Formula: see text] traveled in the bloodstream by pre-supplementing with a traditional Chinese medicine (TCM) Ganoderma tsugae DMSO extract (GTDE) prior to PM[Formula: see text] exposure. Our results show that PM[Formula: see text] caused alterations in enzyme activities and cellular anti-oxidant balance. We found decreased glutathione levels, a reduced cellular redox ratio, increased ROS generation and cytotoxicity in the cellular fractions. The oxidative stress caused DNA damage and apoptosis, likely causing downstream molecular events that trigger vasculature permeabilization and, eventually, cardiovascular disorders. Our results show long-term GTDE treatment increased endogenous glutathione level, while PM[Formula: see text]-reduced glutathione levels and the cellular redox ratio. GTDE was protective against the genotoxic and apoptotic effects initiated by PM[Formula: see text] oxidative stress. Vascular permeability revealed that PM[Formula: see text] only accumulated on the surface of cells after GTDE treatment; no penetration was detected. After two weeks of GTDE treatment, VEGFA secretion was significantly reduced in human umbilical vein endothelial cells (HUVEC) and endothelial cell migration was blocked. Our results suggest GTDE prevents PM[Formula: see text] transmigration into the bloodstream, and the resultant dysfunction, by inhibiting oxidative stress production and endothelial permeability.

Tropomyosin-1 protects transformed alveolar epithelial cells against cigaret smoke extract through the stabilization of F-actin-dependent cell-cell junctions.

The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract.

Xihuang Pill () induces mesenchymal-epithelial transition and inhibits loss of apical-basal polarity in colorectal cancer cell through regulating ZEB1-SCRIB loop.

OBJECTIVE: To investigate the antiproliferative and anti-metastasis effect of Xihuang Pill (, XP) on human colorectal cancer cell and to explore the molecular mechanism by which it produces the effects. METHODS: Highly metastatic human colorectal cancer cell line LoVo was treated with low-, medium-, and highdose XP-containing serum (XP-L, XP-M, XP-H) groups for 48 h, cells intervened with no drug rat serum and PD98059 [extracellular signal-regulated kinase (ERK) inhibitor] as negative and positive controls (NC and PC) groups. Cell proliferation assay was made using cell counting kit-8 (CCK8). The 8 µm pore-size transwell chamber and 4', 6-diamidino-2-phenylindole (DAPI) staining were applied to examine the ability of invasion and migration of the cells. The protein expression of ERK1/2, zinc fifi nger E-box-binding homeobox 1 (ZEB1), Scrib and lethal giant larvae homolog 2 (Lgl2) was detected by Western blotting while the relative mRNA quantity of E-cadherin, N-cadherin, Occludin and junctional adhesion molecule-1 (JAM1) was measured by realtime fluorescent quantitative polymerase chain reaction (RT-qPCR). RESULTS: XP induced a dose-dependent suppression on the proliferation of LoVo cells (P <0.05 or P<0.01), with the inhibition rates varied from 27.30% to 31.08%. Transwell assay showed that when preprocessed with PD98059 and XP-containing serum, the number of cells that passed the filter decreased significantly compared with that of NC group (P <0.05 or P<0.01). Moreover, XP inhibited the protein expression of ERK1/2 and ZEB1 (P <0.05); and up-regulated the protein expression of Scrib and Lgl2 (P <0.05). The mRNA levels of E-cadherin, Occludin and JAM1 of the XP intervened groups and PC group markedly ascended (P <0.05) while that of N-cadherin showed a descending tendency (P>0.05). CONCLUSION: XP intervention suppressed the ability of proliferation, invasion and migration of the LoVo cells. Regulating ZEB1-SCRIB Loop so as to recover epithelial phenotype and apical junctional complex might be one of the mechanisms by which XP produces the anti-metastasis effect.

Estrogen supports urothelial defense mechanisms.

Epidemiological data imply a role of estrogen in the pathogenesis of urinary tract infections (UTIs), although the underlying mechanisms are not well understood. However, it is thought that estrogen supplementation after menopause decreases the risk of recurrent infections. We sought to investigate the influence of estrogen on host-pathogen interactions and the consequences for UTI pathogenesis. We analyzed urothelial cells from menstruating and postmenopausal women before and after a 2-week period of estrogen supplementation, and also studied the influence of estradiol during Escherichia coli UTI in a mouse infection model. Important findings were confirmed in two human urothelial cell lines. We identified two epithelial defense mechanisms modulated by estrogen. Estrogen induced the expression of antimicrobial peptides, thereby enhancing the antimicrobial capacity of the urothelium and restricting bacterial multiplication. In addition, estrogen promoted the expression and redistribution of cell-cell contact-associated proteins, thereby strengthening the epithelial integrity and preventing excessive loss of superficial cells during infection. These two effects together may prevent bacteria from reaching deeper layers of the urinary tract epithelium and developing reservoirs that can serve as a source for recurrent infections. Thus, this study presents some underlying mechanisms for the beneficial effect of estradiol after menopause and supports the application of estrogen in postmenopausal women suffering from recurrent UTI.

Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix.

PURPOSE: This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. METHODS: Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. RESULTS: Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻6 and (3.2 ± 0.3) × 10⁻6 cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 µL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 µM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. CONCLUSIONS: Dietary indicaxanthin and betanin can substantially be absorbed through paracellular junctions of intestinal epithelial cells. Additional trans-membrane permeation can be considered for betanin, whose absorption is limited by a MRP2-mediated efflux and negatively affected by its food matrix. Present findings are consistent with the quite higher bioavailability of indicaxanthin over betanin established in humans.

Phospholipid scramblase 1 is secreted by a lipid raft-dependent pathway and interacts with the extracellular matrix protein 1 in the dermal epidermal junction zone of human skin.

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.

Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway.

Claudin-16 (CLDN-16) is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. The tight junctional localization and Mg(2+) transport of CLDN-16 are regulated by cAMP/PKA-dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN-16 in a direct or indirect manner. CLDN-16 was stably expressed in Madin-Darby canine kidney (MDCK) cells using a Tet-OFF system. The phosphorylation of CLDN-16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN-16 in a concentration-dependent manner. The phosphorylated CLDN-16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg(2+). Vasodilator-stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione-S-transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN-16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN-16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser160Ala) of VASP, CLDN-16 was phosphorylated by DBcAMP and was associated with ZO-1, a tight junctional-scaffolding protein, without integral cell-cell junctions. We suggest that PKA directly phosphorylates CLDN-16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg(2+) reabsorption.

Domain-specific mechanosensory transmission of osmotic and enzymatic cell wall disturbances to the actin cytoskeleton.

Plant protoplasts are embedded within surrounding cell walls and the cell wall-plasma membrane-cytoskeleton (WMC) structural continuum seems to be crucial for the proper functioning of plant cells. We have utilised the protoplast preparation methodology to study the organisation and the putative components of the WMC continuum. Application of an osmotic agent evoked plasmolysis of the Zea mays root apex cells which appeared to be cell type- and growth stage-specific. Simultaneous use of wall polysaccharide-digesting enzymes selectively severed linkages between the components of the WMC continuum which changed the plasmolytic patterns in various cell types. This was followed by a reorganisation of filamentous actin aimed to reinforce protoplast boundaries and maintain the functioning of intercellular contact sites, especially at the cross walls. Particularly strong effects were evoked by pectin-degrading enzymes. Such treatments demonstrated directly the differentiated composition of various wall domains surrounding individual cells with the pectin-enriched cross walls (synapses), and the cellulose-hemicellulose network dominating the side walls. The same wall-degrading enzymes were used for in vitro digestion of isolated Lupinus albus cell walls followed by the extraction of wall proteins. Selective release of proteins suggested the importance of wall polysaccharide-protein interactions in the maintenance of the functioning and mechanical stability of root cell walls.

The focal adhesion kinase amino-terminal domain localises to nuclei and intercellular junctions in HEK 293 and MDCK cells independently of tyrosine 397 and the carboxy-terminal domain.

The function and intracellular localisation of the non-catalytic NH(2)-terminal region of focal adhesion kinase (FAK) are unclear. We investigated the targetting of the FAK NH(2)-terminal domain in HEK 293 and epithelial MDCK cells. Exogenous expression of a variety of GFP-fused and epitope-tagged NH(2) terminal domain constructs either including or lacking the major Tyr 397 autophosphorylation and Src-binding site targeted to nuclei and cell-cell junctions in HEK 293 cells and co-localised at junctions with occludin, and beta1 integrin subunits at junctions. Mutation of Tyr 397 also had no effect on localisation of the NH(2)-terminal domain. In contrast, constructs encoding either the kinase or focal adhesion targeting (FAT) domains but lacking the NH(2)-terminal region failed to localise to intercellular junctions or nuclei. The NH(2)-terminal domain was not associated with beta1 integrin subunits as indicated by co-immunoprecipitation experiments, but did co-localise with cortical actin filaments. The NH(2)-terminal domain also targetted to nuclei and intercellular junctions in MDCK cells, whereas full-length FAK localised only to focal adhesions in these cells. These results indicate that the FAK NH(2)-terminal domain targets to epithelial intercellular junctions and nuclei and suggest novel functions for FAK NH(2)-terminal domain fragments independent of Y397, kinase, and FAT domains.

Transient disruption of intercellular junctions enables baculovirus entry into nondividing hepatocytes.

Baculovirus infection has extended the capabilities for transfection of exogenous genes into a variety of mammalian cell types. Because rat hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented chemically defined medium are an excellent model system for studying liver function in vitro, we investigated the ability of baculoviruses to infect and deliver exogenous genes to cells in this culture system. Efficient delivery to hepatocytes in short-term culture becomes restricted to peripheral cells, or "edge" cells, as the hepatocytes acquire intercellular junctions and form islands with time in culture. This barrier to baculovirus entry can be overcome, and the percentage of internal cells within the hepatocyte islands that are infected with the baculovirus can be increased more than 100-fold, when cells are subjected to transient calcium depletion before and during infection. These findings suggest that at least in some cell types, such as hepatocytes, baculovirus entry may require contact with the basolateral surface. We conclude from this study that recombinant baculovirus infection following transient depletion of extracellular calcium results in delivery of exogenous genes to at least 75% of hepatocytes in long-term DMSO culture, thereby making it possible for the first time to carry out gain-of-function and loss-of-function studies in this cell system.

Localization of a myosin-like protein to plasmodesmata.

Myosin has been localized to plasmodesmata in root tissues of Allium cepa, Zea mays and Hordeum vulgare using a polyclonal antibody to animal myosin in both fluorescence and electron microscopy. Labelling was also observed throughout the cytoplasm, mainly associated with the endoplasmic reticulum and plasma membrane. On Western blots, bands of 180 and 110 kDa were consistently labelled in all three species. These bands were also labelled when the blot was incubated in actin prior to staining with antibodies to actin, raising the possibility that either of these proteins (180 kDa or 110 kDa) may be present in plasmodesmata. Pre-treatment of the tissue with 2,3-butanedione monoxime (BDM), an inhibitor of actin-myosin motility, resulted in a strong constriction of the neck region of plasmodesmata. These results indicate that a myosin-like protein may be present in plasmodesmata and may also play a role in the regulation of transport at the neck region.

Molecular cloning and characterization of human non-smooth muscle calponin.

cDNA clones encoding a calponin isoform with 309 amino acids have been isolated from human heart. The deduced amino acid polypeptide (M(r) 33,697) showed a neutral isoelectric point of 7.1. The mRNA, expressed in cultured smooth muscle cells as well as in fibroblasts, vascular endothelial cells, and keratinocytes, contains a 3' untranslated region of 1.2 kilobases that includes an Alu repetitive sequence in the antisense direction. On the basis of the nucleotide sequence identity to an expressed sequence tag, HUM21ES93 [Cheng, J.-F., Boyartchuk, V., and Zhu, Y. (1994) Genomics 23, 75-84], the human neutral calponin gene is assigned to chromosome 21q11.1. The amino acid sequence indicates that this protein is the human equivalent of mouse calponin-h2 (94.8% identity) [Strasser, P., Gimona, M., Moessler, H., Herzog, M., and Small, J.V. (1993) FEBS Lett. 330, 13-18]. Three tandem repeats of 29 amino acids, a Vav-homologous region and an actin-binding sequence, originally identified in the basic calponin isoform, are conserved. There are two consensus phosphorylation sites for tyrosine kinase. An immunoreactive form of the neutral calponin appears to be localized with vinculin in the cell-to-cell junctions of cardiomyocytes. Mouse calponin-h2 is also expressed in both embryonic and adult heart. These results indicate that the human neutral calponin is a non-smooth muscle isoform, and may play a physiological role in cytoskeletal organization.