Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium

Vascular endothelial-cadherin (VE-cadherin) is a component of the adherens junctions of endothelial cells whose role in endothelial transmigration of leukocytes has been controversial. Using a VE-cadherin/green fluorescent protein fusion construct (VEcadGFP) that mimics the native molecule, we visualized alterations in endothelial junctional structure in real time during transmigration of human neutrophils and monocytes in an in vitro flow model. We observed abundant transmigration occurring exclusively at the cell borders (paracellularly). Surprisingly, transmigration occurred both through de novo formation of transient gaps in VEcadGFP junctional distribution, and also through preexisting gaps. De novo gaps 4-6 microm in size were formed after a leukocyte arrived at a junction, whereas preexisting gaps were present even before the leukocyte had interacted with the endothelial cells contributing to a junction. Gaps rapidly resealed within 5 min after leukocyte transmigration. Migrating leukocytes appeared to push aside VEcadGFP in the plane of the junction, and this displaced material subsequently diffused back to refill the junction. To our knowledge, this is the first example where molecular events at the lateral junction have been tracked in real time during transmigration.     

Thrombin-induced increase in albumin permeability across the endothelium

We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium. The basis for the increased albumin permeability following the addition of alpha-thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps.     

Live imaging of lymphatic development in the zebrafish

The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes. Progress in understanding the origins and early development of this system, however, has been hampered by difficulties in observing lymphatic cells in vivo and in performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. Here, we show that the optically clear developing zebrafish provides a useful model for imaging and studying lymphatic development, with a lymphatic system that shares many of the morphological, molecular and functional characteristics of the lymphatic vessels found in other vertebrates. Using two-photon time-lapse imaging of transgenic zebrafish, we trace the migration and lineage of individual cells incorporating into the lymphatic endothelium. Our results show lymphatic endothelial cells of the thoracic duct arise from primitive veins through a novel and unexpected pathway.     

Ultrastructural distribution of terbium across capillary endothelium: detection by electron spectroscopic imaging and electron energy loss spectroscopy

We used terbium as an intravital tracer of permeability pathways across the walls of capillaries in the rete mirabile of the eel swimbladder and in frog mesentery. Terbium was detected in unstained ultra-thin sections by electron density using electron spectroscopic imaging (ESI) and by electron energy loss spectroscopy (EELS). Enhancement of intrinsic contrast in zero loss images (elastically scattered electrons) permitted imaging of membrane-bound compartments and terbium within them which might otherwise have been undetected in counterstained sections. Element-selective imaging with EELS indicated that terbium was associated with heavy electron-dense deposits, but the terbium mass:volume of sections in areas of lighter deposition was insufficient to obtain a terbium signal. In the rete capillaries, terbium was deposited on the luminal surface, throughout vesicular profiles, and in the interstitium, but could not be traced through interendothelial junctions. Fine terbium deposits were detectable throughout apparent vesicular connections across the endothelium. In the frog mesentery, terbium penetrated some but not all interendothelial clefts, and was detectable in small quantities within luminal and abluminal vesicular profiles and in the interstitium. The results indicate that in the rete capillaries, terbium permeates the capillary via a transcellular route. This route may be provided by transient fusions of luminal and abluminal vesicular compartments.     

Renal metallothionein responds rapidly and site specifically to zinc repletion in growing rats

Metallothionein (MT) is important for heavy metals and free radical protection in the kidney. MT is responsive to zinc and primarily localized within the renal cortex. However, site-specific renal responses to dietary zinc repletion are understudied. The objective of this study was to examine the effects of dietary zinc deficiency and repletion on renal MT concentration and immunolocalization in rats. Weanling male Sprague Dawley rats were randomly assigned to either a zinc-deficient, zinc control, or pair-fed to zinc-deficient group. Half of the zinc-deficient and pair-fed rats were repleted with the control diet ad libitum for an additional 24 h. Renal tissue samples were assessed for total zinc, MT concentrations and MT immunostaining. Dietary zinc deficiency reduced renal zinc and MT concentrations, and attenuated intensity and localization of MT. Dietary zinc repletion for 24 h restored renal zinc and MT concentrations, the latter primarily in the proximal convoluted tubules of the cortex. Concentrations of renal MT, but not zinc, were elevated by diet restriction and MT (μg/mg protein) and partially normalized by 24 h diet repletion. In conclusion, renal MT modification due to zinc deficiency or diet restriction can be rapidly normalized in a site-specific manner with normal dietary zinc intake. The results support a role for MT in kidney homeostasis, in particular at the level of the proximal tubules in the cortex. The speed of MT repletion may have clinical implications for dietary zinc in the treatment of acute and chronic renal pathology due to toxins and free radicals.     

On the mechanism of fluid transport across corneal endothelium and epithelia in general

The mechanism by which fluid is transported across epithelial layers is still unclear. The prevalent idea is that fluid traverses these layers transcellularly, driven by local osmotic gradients secondary to electrolyte transport and utilizing the high osmotic permeability of aquaporins. However, recent findings that some aquaporin knockout mice epithelia transport fluid sow doubts on local osmosis. This review discusses recent evidence in corneal endothelium that points instead to electro-osmosis as the mechanism underlying fluid transport. In this concept, a local recirculating electrical current would result in electro-osmotic coupling at the level of the intercellular junctions, dragging fluid via the paracellular route. The text also mentions possible mechanisms for apical bicarbonate exit from endothelial cells, and discusses whether electro-osmosis could be a general mechanism.     

Skeletal myogenic progenitors in the endothelium of lung and yolk sac

We previously showed that clonable skeletal myogenic cells can be derived from the embryonic aorta but become very rare in the more mature and structured fetal aorta. The aim of this study was to investigate whether, during fetal and postnatal development, these myogenic progenitors progressively disappear or may rather associate with the microvascular district, being thus distributed to virtually all tissues. To test this hypothesis, we used F1 embryos (or mice) from a transgenic line expressing a striated muscle-specific reporter gene (LacZ) crossed with a transgenic line expressing a different endothelial-specific reporter genes (GFP). Endothelial cells were isolated from yolk sac (at E11) and lung (at E11, E17, P1, P10, and P60), two organs embryologically unrelated to paraxial mesoderm, rich in vessels, and devoid of skeletal muscle. Endothelial cells, purified by magnetic bead selection (CD31/PECAM-1(+)) or cell sorting (Tie2-GFP(+)) were then challenged for their skeletal myogenic potential in vitro and in vivo.The results demonstrated that both yolk sac and lung contain progenitor cells, which express endothelial markers and are endowed with a skeletal myogenic potential that they reveal when in the presence of differentiating myoblasts, in vitro, and regenerating muscle, in vivo.The number (or potency to generate skeletal muscle) of these vessels associated cells decreases rapidly with age and is very low in mature animals, possibly correlating with reduced regenerative capacity of adult mammalian tissues.     

Temperature dependence of protein transport across lymphatic endothelium in vitro

The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport. Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies. The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37 degrees C, 36 lymphatics showed a mean rate of protein transport of 3.51 +/- 0.45 (SEM) micrograms/min per cm2 of lymphatic endothelium. The rate was influenced by the temperature of the system, being significantly reduced by 49% +/- 4.8, 31% +/- 5.3, and 29% +/- 3.9 when the temperature was lowered to 4 degrees, 24 degrees, and 30 degrees C, respectively. When the temperature was raised to 40 degrees C, the rate was significantly increased by 48% +/- 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways. No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24 degrees, 30 degrees, and 40 degrees C. At 4 degrees C, the temperature at which the rate of protein transport was maximally reduced, vesicular size decreased, and the number of free cytoplasmic vesicles increased, whereas the number associated with the abluminal and luminal surfaces decreased. We concluded that isolated perfused lymphatic segments transport protein at a relatively constant rate under control conditions, and that this transendothelial transport comprises both temperature-dependent and temperature-independent mechanisms. The findings were considered in terms of the different theories of lymph formation and were interpreted as providing support for the vesicular theory.     

Ionic selectivity of the paracellular shunt path across rabbit corneal endothelium

We have measured the dilution and biionic potentials across the isolated rabbit corneal endothelium in order to learn about the ionic selectivity of its intercellular junctions. Single-salt dilution potentials have been measured as a function of [NaCl] or [NaHCO3] gradients across the tissue. Biionic potentials were similarly measured by replacing Na+ with K+ on either side of the tissue. The potentials thus measured were fit to the constant field equation and to an approximation of it to obtain the ionic permeabilities for K+, HCO-3 and Cl- relative to Na+. The permeability sequence obtained was PK greater than PNa greater than PHCO3 approximately equal to PCl. Potentials were also measured after imposing an osmotic gradient across the preparation using sucrose. The results obtained with all these methods are consistent and suggest that this tissue is slightly more permeant to cations than anions, but that the selectivity of the intercellular junction is relatively low. From these experiments, a 30 mM gradient of salt across the endothelial layer would be needed in order to explain the observed spontaneous potential difference (about 1 mV, aqueous negative) across that layer if the potential was due to the selectivity of the intercellular junctions. Such a value for the gradient is much larger than theoretical estimates of it; therefore, we favor electrogenic transport of HCO-3 as a better explanation for the origin of the spontaneous potential difference.     

Mechanism of the chilling-induced decrease in proton pumping across the tonoplast of rice cells

The ATP-generated proton pumping across tonoplast vesicles from chilling-sensitive Boro rice (Oryza sativa L. var. Boro) cultured cells was markedly decreased by chilling at 5 degrees C for 3 d. The membrane fluidity of core hydrophobic and surface hydrophilic regions of the lipid bilayer was measured by steady-state fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium 1,6-diphenyl-1,3,5-hexatriene and by electron spin resonance spectroscopy of 16- and 5-doxyl stearic acid, respectively. The fluidity of the surface region of the lipid bilayer of the tonoplast vesicles decreased by chilling. The fluidity of the surface region of the liposomes and the proton pumping across the reconstituted proteoliposomes with tonoplast H+-ATPase decreased with increasing content of the glycolipids. The proton pumping across chimera proteoliposomes was reduced by chilling only when it was reconstituted in the presence of tonoplast glycolipids from chilled Boro cells. These data suggest that the reduction in ATP-generated proton pumping across the tonoplast by chilling is due to the decrease in the fluidity of the surface region of the lipid bilayer of the tonoplast, which is caused by the changes in glycolipids.     

Analysis of presteady-state Na+ fluxes across the rabbit corneal endothelium

Summary Instead of the conventional steady-state fluxes, the presteady-state fluxes of²²Na across the rabbit corneal endothelium were measured. In contrast to reports that there is no net Na⁺ movement across the corneal endothelium, we find a net transport of Na⁺ across this tissue. The direction of net Na⁺ flux is from the stromal to the aqueous side and the magnitude is 2.3±0.4 eq/cm²·hr (n=11,sem). Net Na⁺ transport is inhibited in the presence of ouabain (10^–4 m). Acetazolamide (10^–4 m) has only a slight inhibitory effect on the rate of Na⁺ transport but decreases the transendothelial potential difference by about 30%. The passive component of the Na⁺ transport has been estimated by analyzing the presteady-state influx and efflux curves and found to occur 10% via cellular and 90% via paracellular routes. The analysis for the separation of the pathways has been based on a recently proposed theory which holds that the flux ratio, regardless of its driving forces, is independent of time.     

Live cell imaging of the HIV-1 life cycle

Technology developed in the past 10 years has dramatically increased the ability of researchers to directly visualize and measure various stages of the HIV type 1 (HIV-1) life cycle. In many cases, imaging-based approaches have filled critical gaps in our understanding of how certain aspects of viral replication occur in cells. Specifically, live cell imaging has allowed a better understanding of dynamic, transient events that occur during HIV-1 replication, including the steps involved in viral fusion, trafficking of the viral nucleoprotein complex in the cytoplasm and even the nucleus during infection and the formation of new virions from an infected cell. In this review, we discuss how researchers have exploited fluorescent microscopy methodologies to observe and quantify these events occurring during the replication of HIV-1 in living cells.     

特异性肝癌细胞结合单链抗体的筛选及克隆表达

用人肝癌细胞系SMMC7721免疫小鼠,提取脾细胞总RNA,构建单链抗体库,从中筛选到1个与人肝癌细胞系HepG2特异性结合的噬菌体－单链抗体。此单链抗体与HepG2细胞结合滴度比正常肝细胞低100倍以上。构建表达质粒pTrx-scFv5-56,单链抗体scFv5-56在大肠杆菌BL21（DE3）中成功地进行了可溶性表达。     

An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii

Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy.     

Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on I(Cl,swell), the swelling-induced Cl- current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect I(Cl,swell). However, transfection of c-Src with a Ser3Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotonicity-induced I(Cl,swell) in CPAE cells. Kinase activity was dispensable for the inhibition of I(Cl,swell), since kinase-deficient c-Src Ser3Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the Ser3Cys mutation was required to obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition.     

Construction and expression of antibody targeted plasminogen activator*

It has been known that antibody-mediated plasminogen activator will be much more specific than its parent molecular. To get a cheaper and more effective medicine for thrombolytic therapy, we used SZ51, a GMP140 specific monoclonal antibody, and a truncated single-chain urokinase to construct a novel targeted plasminogen activator. PCR was used to amplify the region of VL and VH chains from Fab of SZ51, GMP140 specific monoclonal antibody, and scu-PA-32KD(leu144-leu411) from urokinase gene, respectively. Through suitable linker and appropriate restriction sites, these fragments were joined together and inserted into the expression vector, pET-5a, via NdeI site. The recombinant protein was expressed in BL21 (DE3) plyS, a kind of E. coli. It was shown in Western-blotting and ELISA that the protein could interact with the multiple cloned antibody of urokinase. After partial purification: dialysis, Sephadex G-100, dialysis and Phenyl-Sepharose fast flow, the product had a strong fibrinolytic activity through activating plasminogen on fibrin plate. The specific activity was about 47,000 IU/mg, corresponding to 80,000 IU/mg for the part of rscu-PA-32k, and the activity could be inhibited specifically by urokinase specific antibody. Activation of plasminogen by the chimera followed Michaelis-Menten kinetics, and the K_m was 1.08 uM.     

Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.     

Live imaging of mitosomes and hydrogenosomes by HaloTag technology

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes.