Endothelial hyperpermeability is a significant problem in vascular inflammation associated with trauma, ischaemia-reperfusion injury, sepsis, adult respiratory distress syndrome, diabetes, thrombosis and cancer. An important mechanism underlying this process is increased paracellular leakage of plasma fluid and protein. Inflammatory stimuli such as histamine, thrombin, vascular endothelial growth factor and activated neutrophils can cause dissociation of cell-cell junctions between endothelial cells as well as cytoskeleton contraction, leading to a widened intercellular space that facilitates transendothelial flux. Such structural changes initiate with agonist-receptor binding, followed by activation of intracellular signalling molecules including calcium, protein kinase C, tyrosine kinases, myosin light chain kinase, and small Rho-GTPases; these kinases and GTPases then phosphorylate or alter the conformation of different subcellular components that control cell-cell adhesion, resulting in paracellular hypermeability. Targeting key signalling molecules that mediate endothelial-junction-cytoskeleton dissociation demonstrates a therapeutic potential to improve vascular barrier function during inflammatory injury.Endothelial cells lining the inner surface of microvessels form a semipermeable barrier that actively participates in blood-tissue exchange of plasma fluid, proteins and cells. The precise regulation of endothelial permeability is essential for maintaining circulatory homeostasis and the physiological function of different organs. As a result, microvascular barrier dysfunction and endothelial hyperpermeability represent crucial events in the development of a variety of disease processes, such as adult respiratory distress syndrome (ARDS), ischemia-reperfusion (I-R) injury, diabetic vascular complications, and tumour metastasis. Better insight into the molecular mechanisms underlying pathogenic conditions related to microvascular hyperpermeability is required for developing effective therapeutic strategies. Following intensive studies over the past few decades, it is now understood that endothelial permeability is mediated through a transcellular pathway (across cells) and a paracellular pathway (between cells), both of which are highly regulated by mechanical forces and biochemical signals. Transcellular versus paracellular permeabilityAn important molecular mechanism underlying transcellular permeability is macromolecule transcytosis via caveoli -specialised plasmalemmal vesicles containing caveolin-1. The involvement of caveolin-1 in regulating cardiovascular functions associated with endothelial barrier properties has been demonstrated through studies using transgenic and knockout NIH Public Access Author ManuscriptExpert Rev Mol Med. Author manuscript; available in PMC 2010 February 24. Published in final edited form as:Expert Rev Mol Med. ; 11: e19. doi:10.1017/S1462399409001112. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript animals (Refs 1,2,3,4). Upon binding to...

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In this study we investigated the dynamics of R-Ras intracellular trafficking and its contributions to the unique roles of R-Ras in membrane ruffling and cell spreading. Wild type and constitutively active R-Ras localized to membranes of both Rab11- and transferrin-positive and -negative vesicles, which trafficked anterograde to the leading edge in migrating cells. H-Ras also co-localized with R-Ras in many of these vesicles in the vicinity of the Golgi, but R-Ras and H-Ras vesicles segregated proximal to the leading edge, in a manner dictated by the C-terminal membrane-targeting sequences. These segregated vesicle trafficking patterns corresponded to distinct modes of targeting to membrane ruffles at the leading edge. Geranylgeranylation was required for membrane anchorage of R-Ras, whereas palmitoylation was required for exit from the Golgi in post-Golgi vesicle membranes and trafficking to the plasma membrane. R-Ras vesicle membranes did not contain phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), whereas R-Ras co-localized with PtdIns(3,4,5)P3 in membrane ruffles. Finally, palmitoylation-deficient R-Ras blocked membrane ruffling, R-Ras/PI3-kinase interaction, enrichment of PtdIns(3,4,5)P3 at the plasma membrane, and R-Ras-dependent cell spreading. Thus, lipid modification of R-Ras dictates its vesicle trafficking, targeting to membrane ruffles, and its unique roles in localizing PtdIns(3,4,5)P3 to ruffles and promoting cell spreading.

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Six male Tharparkar cattle aged 2-3 years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15 days and then exposed to 42 °C temperature up to 23 days followed by 12 days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6 h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P < 0.05) and showed two peaks on day 17 and day 32 (2nd and 17th days of thermal challenge, respectively). The HSP70 mRNA expression was increased (P < 0.05) in a temperature- and time-dependent manner in heat stress challenge treatment as compared to control in cultured PBMCs. HSP70 expression was found to be higher (P < 0.05) after 10 days of heat exposure (corresponds to chronic heat stress) as compared to the first 5 days of heat stress (corresponds to short-term heat stress) and control period at TNZ. The present findings indicate that HSP70 is possibly involved in heat stress adaptive response in Tharparkar cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

Menacing global rise in surface temperature compelled more focus of research over understanding heat stress response mechanism of animals and mitigation of heat stress. Twenty-four goats divided into four groups (n = 6) such as NHS (non-heat-stressed), HS (heat-stressed), HS + VC (heat-stressed administered with vitamin C), and HS + VE + Se (heat-stressed administered with vitamin E and selenium). Except NHS group, other groups were exposed to repeated heat stress (42 °C) for 6 h on 16 consecutive days. Blood samples were collected at the end of heat exposure on days 1, 6, 11, and 16. When groups compared between days, expression of all heat shock proteins (HSPs) showed a similar pattern as first peak on day 1, reached to basal level on the sixth day, and followed by second peak on day 16. The relative messenger RNA (mRNA) and protein expression of HSP 60, HSP70, and HSP90 was observed highest (P < 0.05) in HS group, followed by antioxidant-administered group on days 1 and 16, which signifies that antioxidants have dampening effect on HSP expression. HSP105/110 expression was highest (P < 0.05) on day 16. We conclude that HSP expression pattern is at least two-peak phenomenon, i.e., primary window of HSP protection on the first day followed by second window of protection on day 16. HSP60, HSP70, and HSP90 play an important role during the initial phase of heat stress acclimation whereas HSP105/110 joins this cascade at later phase. Antioxidants may possibly attenuate the HSP expression by reducing the oxidative stress.

Six, nonpregnant, Barbari goats aged 4-5 years were selected for the study. For the first 6 days, the animals were kept in psychrometric chamber at thermoneutral temperature for 6 h each day to make them acclimated to climatic chamber. On the 7th day, the animals were exposed to 41 °C temperature for 3 h and then to 45 °C for the next 3 h. Cardinal physiological responses were measured, and blood samples (3 ml) were collected at 1-h interval during the heat exposure period and then once after 6 h of the heat exposure. The rectal temperature (RT) and respiratory rate (RR) increased significantly (P < 0.05) during the heat exposure compared to pre- and postexposure. The relative messenger RNA (mRNA) expression of heat shock protein (HSP)60, HSP70, and HSP90 increased significantly (P < 0.05) within 1 h after exposure to heat stress at 41 and 45 °C and decreased significantly (P < 0.05) in next 2 h but remain significantly (P < 0.05) elevated from preexposure. HSP105/110 relative mRNA expression level remained unchanged during the first 4 h, and thereafter, it increased significantly (P < 0.05) and reached the peak at 6 h. Relative protein expression pattern of HSPs during exposure to heat stress showed similar trend as observed for the relative mRNA expression. Given the response sensitivity and intensity of HSP genes to environmental stresses, HSP70 was found to be the most sensitive to temperature fluctuation, and it could be used as an important molecular biomarker to heat stress in animals.