International trade standards for commodities and products derived from animals: the need for a system that integrates food safety and animal disease risk management.

A case is made for greater emphasis to be placed on value chain management as an alternative to geographically based disease risk mitigation for trade in commodities and products derived from animals. The geographic approach is dependent upon achievement of freedom in countries or zones from infectious agents that cause so-called transboundary animal diseases, while value chain-based risk management depends upon mitigation of animal disease hazards potentially associated with specific commodities or products irrespective of the locality of production. This commodity-specific approach is founded on the same principles upon which international food safety standards are based, viz. hazard analysis critical control points (HACCP). Broader acceptance of a value chain approach enables animal disease risk management to be combined with food safety management by the integration of commodity-based trade and HACCP methodologies and thereby facilitates 'farm to fork' quality assurance. The latter is increasingly recognized as indispensable to food safety assurance and is therefore a pre-condition to safe trade. The biological principles upon which HACCP and commodity-based trade are based are essentially identical, potentially simplifying sanitary control in contrast to current separate international sanitary standards for food safety and animal disease risks that are difficult to reconcile. A value chain approach would not only enable more effective integration of food safety and animal disease risk management of foodstuffs derived from animals but would also ameliorate adverse environmental and associated socio-economic consequences of current sanitary standards based on the geographic distribution of animal infections. This is especially the case where vast veterinary cordon fencing systems are relied upon to separate livestock and wildlife as is the case in much of southern Africa. A value chain approach would thus be particularly beneficial to under-developed regions of the world such as southern Africa specifically and sub-Saharan Africa more generally where it would reduce incompatibility between attempts to expand and commercialize livestock production and the need to conserve the subcontinent's unparalleled wildlife and wilderness resources.

Balancing livestock production and wildlife conservation in and around southern Africa's transfrontier conservation areas.

Biodiversity conservation, of which the transfrontier conservation area movement is an integral part, and more effective livestock production/trade are pivotal to future rural development in southern Africa. For that reason, it is imperative to effectively ameliorate the obstacles that have impeded progress towards the coexistence of these two sectors for more than half a century. Transboundary animal diseases, foot and mouth disease in particular, have been and continue to be the most important of these obstacles. Fortunately, new developments in international sanitary standards applicable to trade in commodities and products derived from animals are beginning to make a solution possible. However, while progress in principle has been achieved, practical implementation remains problematic for technical reasons, exacerbated by inconsistent attitudes towards acceptance of non-traditional international trade standards. This paper describes the background to this situation, progress that has been achieved in the recent past and remaining difficulties that need to be overcome to advance towards achievement of balanced rural development in southern Africa.

Effects of meloxicam or tolfenamic acid administration on the pain and stress responses of Merino lambs to mulesing.

OBJECTIVE: To determine the effectiveness of two long-acting non-steroidal anti-inflammatory drugs (NSAIDs) at reducing the pain and stress responses to mulesing in lambs. PROCEDURES: Merino lambs (n = 60) were allocated at 5 weeks of age to six treatment groups: (1) sham mules; (2) mules; (3) tolfenamic acid-sham mules; (4) tolfenamic acid administered 45 min before mulesing; (5) tolfenamic acid at the time of mulesing; (6) meloxicam at the time of mulesing. Plasma cortisol was measured at -0.75, -0.25, 0, 0.5, 1, 3, 6, 12, 24, 48 and 72 h relative to mulesing. Beta-endorphin concentrations in plasma were determined at 0, 0.5, 1, 6, 12, 24, and 48 h. Haematology was performed on blood samples taken at -0.75, 0, 24, 48 and 72 h. Plasma haptoglobin was measured at 0, 12, 24, 48 and 72 h. Rate of wound healing was determined 72 h post mulesing, and animal behaviour, including posture, was measured for 6 h after mulesing. RESULTS: The mulesed lambs exhibited large increases in plasma concentrations of cortisol, beta-endorphin and haptoglobin. All mulesed animals lost weight significantly in the week after mulesing, regardless of analgesic administration, but the difference in weight between mulesed and unmulesed lambs was less at the final measurement, 2 weeks after mulesing. Mulesed lambs spent significantly less time lying ventrally than control lambs. All lambs that were mulesed, including those administered NSAIDs, spent more time standing with a hunched posture and less time walking normally than control lambs. CONCLUSIONS: The NSAID treatments applied 45 min before or at the time of mulesing at the dose levels used in this study were not effective in reducing the acute response of lambs to mulesing.

The effect of a topical anaesthetic formulation, systemic flunixin and carprofen, singly or in combination, on cortisol and behavioural responses of Merino lambs to mulesing.

OBJECTIVE: To determine the pain responses of lambs to mulesing, and the effectiveness of potential analgesic treatments. PROCEDURES: Merino lambs (n=64) were allocated at 5 weeks of age to eight treatment groups: 1) sham mules; 2) conventional mules; 3) topical anaesthetic, incorporating lignocaine, bupivicaine, adrenaline and cetrimide, applied immediately after mulesing; 4) flunixin + topical anaesthetic, with flunixin administered 2.5 mg/kg s.c. 90 min before mulesing; 5) carprofen + topical anaesthetic, with carprofen administered 4 mg/kg s.c. 90 min before mulesing; 6) carprofen, administered as above; 7) flunixin, administered as above; and 8) carprofen + flunixin, administered as above. Plasma cortisol was measured at 0, 0.5, 6, 12 and 24 h relative to mulesing. Animal behaviour, including posture, was recorded for 12 h after mulesing. RESULTS: The conventional mules lambs exhibited large increases in plasma cortisol, reduced lying and increased standing with a hunched back compared with sham mules animals. Topical anaesthetic reduced the cortisol peak to mulesing and hunched standing, and increased lying compared with the conventional mules treatment, but generally did not result in values equivalent to sham mules animals. Carprofen, flunixin, and carprofen + flunixin treatments did not reduce the cortisol response to mulesing but substantially ameliorated some changes in behavioural postures. Flunixin + topical anaesthetic reduced the cortisol peak following mulesing and substantially ameliorated most changes in behavioural postures. Carprofen + topical anaesthetic abolished the cortisol peak following mulesing and substantially ameliorated most changes in behavioural postures. All mulesed animals lost weight in the week after mulesing regardless of analgesic administration, but there were no significant differences in growth rate between any of the eight treatments over the 3 weeks after mulesing. CONCLUSIONS: Analgesics can moderate the pain response of lambs to mulesing. The welfare outcome for lambs of mulesing could be improved by use of a combination of local anaesthetic and long acting non-steroidal anti-inflammatory drug.

Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion.

Disruption of the actin cytoskeleton in proximal tubule cells is a key pathophysiological factor in acute renal failure. To investigate dynamic alterations of the actin cytoskeleton in live proximal tubule cells, LLC-PK(10) cells were transfected with an enhanced yellow fluorescence protein (EYFP)-actin construct, and a clone with stable EYFP-actin expression was established. Confluent live cells were studied by confocal microscopy under physiological conditions or during ATP depletion of up to 60 min. Immunoblots of stable transfected LLC-PK(10) cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogeneous cytosolic distribution of EYFP-actin indicated colocalization with G-actin as well. Beyond previous findings, we observed differential subcellular disassembly of F-actin structures: stress fibers tagged with EYFP-actin underwent rapid and complete disruption, whereas cortical and microvillar actin disassembled at slower rates. In parallel, ATP depletion induced the formation of perinuclear EYFP-actin aggregates that colocalized with F-actin. During ATP depletion the G-actin fraction of EYFP-actin substantially decreased while endogenous and EYFP-F-actin increased. During intracellular ATP repletion, after 30 min of ATP depletion, there was a high degree of agreement between F-actin formation from EYFP-actin and endogenous actin. Our data indicate that EYFP-actin did not alter the characteristics of the endogenous actin cytoskeleton or the morphology of LLC-PK(10) cells. Furthermore, EYFP-actin is a suitable probe to study the spatial and temporal dynamics of actin cytoskeleton alterations in live proximal tubule cells during ATP depletion and ATP repletion.

Rho-kinase regulates myosin II activation in MDCK cells during recovery after ATP depletion.

Alterations in the actin cytoskeleton of renal tubular epithelial cells during periods of ischemic injury and recovery have important consequences for normal cell and kidney function. Myosin II has been demonstrated to be an important effector in organizing basal actin structures in some cell types. ATP depletion in vitro has been demonstrated to recapitulate alterations of the actin cytoskeleton in renal tubular epithelial cells observed during renal ischemia in vivo. We utilized this reversible cell culture model of ischemia to examine the correlation of the activation state and cellular distribution of myosin II with disruption of actin stress fibers in Madin-Darby canine kidney (MDCK) cells during ATP depletion and recovery from ATP depletion. We found that myosin II inactivation occurs rapidly and precedes dissociation of myosin II from actin stress fibers during ATP depletion. Myosin II activation temporally correlates with colocalization of myosin II to reorganizing stress fibers during recovery from ATP depletion. Furthermore, myosin activation and actin stress fiber formation were found to be Rho-associated Ser/Thr protein kinase dependent during recovery from ATP depletion.

Specific collagenolysis by gelatinase A, MMP-2, is determined by the hemopexin domain and not the fibronectin-like domain.

In view of the essential role of the hemopexin domain of the traditional interstitial collagenases, MMP-1, -8, -13 and MT1-MMP (MMP-14), in determining specific collagen cleavage we have studied the function of this domain in MMP-2, relative to that of the fibronectin-like domain that promotes gelatinolysis. Although the fibronectin-like domain promotes avid binding to collagen, our data demonstrate that the catalytic and hemopexin domains of MMP-2 are sufficient to effect the critical step in cleavage of rat type I collagen into 3/4 and 1/4 fragments. The mechanism of MMP-2 cleavage of collagen proceeds in two phases, the first resembling that of the interstitial collagenases, followed by gelatinolysis, promoted by the fibronectin-like domain.

Myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1.

Activation of myosin II by myosin light chain kinase (MLCK) produces the force for many cellular processes including muscle contraction, mitosis, migration, and other cellular shape changes. The results of this study show that inhibition or potentiation of myosin II activation via over-expression of a dominant negative or wild type MLCK can delay or accelerate tumor necrosis factor-alpha (TNF)-induced apoptotic cell death in cells. Changes in the activation of caspase-8 that parallel changes in regulatory light chain phosphorylation levels reveal that myosin II motor activities regulate TNF receptor-1 (TNFR-1) signaling at an early step in the TNF death signaling pathway. Treatment of cells with either ionomycin or endotoxin (lipopolysaccharide) leads to activation of myosin II and increased translocation of TNFR-1 to the plasma membrane independent of TNF signaling. The results of these studies establish a new role for myosin II motor activity in regulating TNFR-1-mediated apoptosis through the translocation of TNFR-1 to or within the plasma membrane.

Perivascular cells regulate endothelial membrane type-1 matrix metalloproteinase activity.

Angiogenic stimuli selectively induced expression of membrane type-1 matrix metalloproteinase (MT1-MMP) transcripts and protein in human umbilical vein endothelial cells (HUVECs). Pro-MMP-2 activation was blocked by treatment with tissue inhibitor of metalloproteinases-2 (TIMP-2), but not by TIMP-1 or inhibitors of other proteinase classes. Anti-MT1-MMP antibodies abrogated recombinant pro-MMP-2 activation by plasma membranes, indicating that MT1-MMP is the main mediator of pro-MMP-2 activation in HUVECs. Cocultures of HUVECs with smooth muscle cells (SMC) or pericytes (PC) resulted in the suppression of HUVEC pro-MMP-2 activation. Treatment of A10 SMC conditioned media with a neutralising anti-TIMP-2 antibody prevented the suppression of HUVEC pro-MMP-2 activation. Inhibition of HUVEC MT1-MMP function by PC and SM3 SMC correlated with elevated TIMP-3 expression. Thus, perivascular supporting cells regulate the functions of proangiogenic MMPs elaborated by endothelial cells via selective expression of TIMPs. This interplay may be important for maintenance of blood vessel architecture and neovascularisation.

Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species.

Thrombin, a critical enzyme in the coagulation cascade, has also been associated with angiogenesis and activation of the zymogen form of matrix metalloproteinase-2 (MMP-2 or gelatinase-A). We show that thrombin activated pro-MMP-2 in a dose- and time-dependent manner in cultured human umbilical-vein endothelial cells (HUVECs) to generate a catalytically active 63 kDa protein that accumulated as the predominant form in the conditioned medium. This 63 kDa thrombin-activated MMP-2 is distinct from the 62 kDa species found following concanavalin A or PMA stimulated pro-MMP-2 activation. Hirudin and leupeptin blocked thrombin-induced pro-MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, since it was blocked by EDTA, o-phenanthroline, hydroxamate metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-4, but not TIMP-1. Thrombin inefficiently cleaved recombinant 72 kDa pro-MMP-2, but efficiently cleaved the 64 kDa MT-MMP-processed intermediate form in the presence of cells. Thrombin also rapidly (within 1 h) increased cellular MT-MMP activity, and at longer time points (>6 h) it increased expression of MT1-MMP mRNA and protein. Thus signalling via proteinase-activated receptors (PARs) may play a role in thrombin-induced MMP-2 activation, though this does not appear to involve PAR1, PAR2, or PAR4 in HUVECs. These results indicate that in HUVECs the activation of pro-MMP-2 by thrombin involves increased MT-MMP activity and preferential cleavage of the MT-MMP-processed 64 kDa MMP-2 form in the presence of cells. The integration of these proteinase systems in the vascular endothelium may be important during thrombogenesis and tissue remodelling associated with neovascularization.

Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization.

Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell-cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.

Membrane type 1 matrix metalloproteinase and gelatinase A synergistically degrade type 1 collagen in a cell model.

A fibrosarcoma cell line transfected with the matrix metalloproteinase MT1 MMP showed an enhanced ability to degrade 14C-labelled collagen films. As previously shown for proMMP 2 activation, TIMP 1 was an ineffective inhibitor of the process of collagenolysis whereas TIMP 2 was efficient and completely prevented collagen degradation. In the presence of the calcium ionophore, ionomycin, proteolytic processing of MT1 MMP was restricted and collagenolysis did not occur indicating that the 63 kDa form of the enzyme is not a functional collagenase. The collagenolytic activity of MT1 MMP was shown to be enhanced by the addition of proMMP 2, but TIMP 1 inhibition remained poor relative to that of TIMP 2. The study demonstrated that synergy between two non-conventional collagenases effectively degrades insoluble pericellular collagen. Due to the membrane localisation of MT1 MMP, this could potentially occur in a highly localised manner.

Dynamics of the Dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis.

The Arp2/3 complex is a ubiquitous and important regulator of the actin cytoskeleton. Here we identify this complex from Dictyostelium and investigate its dynamics in live cells. The predicted sequences of the subunits show a strong homology to the members of the mammalian complex, with the larger subunits generally better conserved than the smaller ones. In the highly motile cells of Dictyostelium, the Arp2/3 complex is rapidly re-distributed to the cytoskeleton in response to external stimuli. Fusions of Arp3 and p41-Arc with GFP reveal that in phagocytosis, macropinocytosis, and chemotaxis the complex is recruited within seconds to sites where actin polymerization is induced. In contrast, there is little or no localization to the cleavage furrow during cytokinesis. Rather the Arp2/3 complex is enriched in ruffles at the polar regions of mitotic cells, which suggests a role in actin polymerization in these ruffles.

Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.

Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G-->A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways. (Blood. 2000;96:1646-1654)

Rho controls actin cytoskeletal assembly in renal epithelial cells during ATP depletion and recovery.

Actin cytoskeletal disruption is a hallmark of ischemic injury and ATP depletion in a number of cell types, including renal epithelial cells. We manipulated Rho GTPase signaling by transfection and microinjection in LLC-PK proximal tubule epithelial cells and observed actin cytoskeletal organization following ATP depletion or recovery by confocal microscopy and quantitative image analysis. ATP depletion resulted in disruption of stress fibers, cortical F-actin, and apical actin bundles. Constitutively active RhoV14 prevented disruption of stress fibers and cortical F-actin during ATP depletion and enhanced the rate of stress fiber reassembly during recovery. Conversely, the Rho inhibitor C3 or dominant negative RhoN19 prevented recovery of F-actin assemblies upon repletion. Actin bundles in the apical microvilli and cytosolic F-actin were not affected by Rho signaling. Assembly of vinculin and paxillin into focal adhesions was disrupted by ATP depletion, and constitutively active RhoV14, although protecting stress fibers from disassembly, did not prevent dispersion of vinculin and paxillin, resulting in uncoupling of stress fiber and focal adhesion assembly. We propose that ATP depletion causes Rho inactivation during ischemia and that recovery of normal cellular architecture and function requires Rho.

Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations.

Apical membrane of renal proximal tubule cells is extremely sensitive to ischemia, with structural alterations occurring within 5 min. These changes are felt secondary to actin cytoskeletal disruption, yet the mechanism responsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-binding protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F-actin binding, depolymerization, and severing, and because ADF activation occurs by dephosphorylation, we questioned whether ADF played a role in microvilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluorescence and Western blot studies on cortical tissue documented the presence of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the Triton X-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylated forms were identified. During ischemia, marked alterations occurred. Intraluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occurring duration-dependent dephosphorylation of ADF. At 0-30 min of ischemia, total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collected under physiological conditions did not contain ADF or actin, whereas urine collected after 30 min of ischemia contained both ADF and actin. Reperfusion was associated with normalization of cellular pADF levels, pADF intracellular distribution, and repair of apical microvilli. These data suggest that activation of ADF during ischemia via dephosphorylation is, in part, responsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles.

Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense.

In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2-/- neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2-/- bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions.

Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation.

Rho family GTPases regulate multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These effects are achieved through the interaction of GTP-bound proteins with various downstream targets. A series of RhoA/Rac1 and Rho/Ras chimeras was generated to map the domain(s) of RhoA involved in its association with two classes of effector kinase, represented by PRK2 and ROCK-I. Although the switch 1 domain was required for effector binding, the N terminus of Rho (residues 1-75) was interchangeable with that of Rac. This suggested that the region of Rho that confers effector binding specificity lay further C-terminal. Subsequent studies indicated that the "insert domain"(residues 123-137), a region unique to Rho family GTPases, is not the specificity determinant. However, a determinant for effector binding was identified between Rho residues 75-92. Rac to Rho point mutations (V85D or A88D) within loop 6 of Rac promoted its association with PRK2 and ROCK, whereas the reciprocal Rho(D87V/D90A) double mutant significantly reduced effector binding capacity. In vivo studies showed that microinjection of Rac(Q6IL/V85D/A88D) but not Rac(Q6IL) induced stress fiber formation in LLC-PK epithelial cells, suggesting that loop 6 residues conferred the ability of Rac to activate ROCK. On the other hand, the reciprocal Rho (Q6IL/D87V/D90A) mutant was defective in its ability to transform NIH 3T3 cells. These data suggest that although Rho effectors can utilize a Rho or Rac switch 1 domain to sense the GTP-bound state of Rho, unique residues within loop 6 are essential for determining both effector binding specificity and cellular function.

Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion.

Tight junctions control paracellular permeability and cell polarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro model of renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rho signaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells than control junctions, whereas cells expressing activated Rho better maintained junctions during ATP depletion than control cells. ATP depletion and Rho signaling altered phosphorylation signaling mechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoamino acid content following ATP depletion, which was restored on recovery. Expression of Rho mutant proteins in MDCK cells also altered levels of occludin serine/threonine phosphorylation, indicating that occludin is a target for Rho signaling. We conclude that Rho GTPase signaling induces posttranslational effects on tight junction components. Our data also demonstrate that activating Rho signaling protects tight junctions from damage during ATP depletion.