CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced endothelial cell migration

The migration of endothelial cells (ECs) plays critical roles in vascular physiology and pathology. The receptors CXCR1 and CXCR2, known as G protein-coupled receptors which are essential for migratory response of ECs toward the shear stress-dependent CXCL8 (interleukin-8), are potential mechano-sensors for mechanotransduction of the hemodynamic forces. In present study, the mRNA and protein expression of CXCR1 and CXCR2 in EA.hy926 cells was detected by RT-PCR and Western blot analysis under three conditions of laminar shear stress (5.56, 10.02 and 15.27 dyn/cm(2)) respectively. Using a scratched-wound assay, the effects of CXCR1 and CXCR2 were assessed by the percentage of wound closure while CXCR1 and CXCR2 were functional blocked by the CXCL8 receptor antibodies. The results showed that the mRNA and protein expression of CXCR1 and CXCR2 was both upregulated by 5.56 dyn/cm(2) laminar shear stress, but was both downregulated by 15.27 dyn/cm(2). The wound closure was inhibited significantly while cells were treated with those antibodies in all the conditions. It was suggested that CXCR1 and CXCR2 are involved in mediating the laminar shear stress-induced EC migration. Taken together, these findings indicated that CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced EC migration. Understanding this expanded mechanism of laminar shear stress-induced cell migration will provide novel molecular targets for therapeutic intervention in cancer and cardiovascular diseases.     

Rac1 mediates laminar shear stress-induced vascular endothelial cell migration

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. ECs are constantly subjected to shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular behaviors and functions. The aim of this study is to elucidate the effects of Rac1, which is the member of small G protein family, on EC migration under different laminar shear stress (5.56, 10.02, and 15.27 dyn/cm²). The cell migration distance under laminar shear stress increased significantly than that under the static culture condition. Especially, under relative high shear stress (15.27 dyn/cm²) there was a higher difference at 8 h (P < 0.01) and 2 h (P < 0.05) compared with static controls. RT-PCR results further showed increasing mRNA expression of Rac1 in ECs exposed to laminar shear stress than that exposed to static culture. Using plasmids encoding the wild-type (WT), an activated mutant (Q61L), and a dominant-negative mutant (T17N), plasmids encoding Rac1 were transfected into EA.hy 926 cells. The average net migration distance of Rac1Q61L group increased significantly, while Rac1T17N group decreased significantly in comparison with the static controls. These results indicated that Rac1 mediated shear stress-induced EC migration. Our findings conduce to elucidate the molecular mechanisms of EC migration induced by shear stress, which is expected to understand the pathophysiological basis of wound healing in health and diseases.     

Moisture adsorption capacity and surface area as deduced from vapour pressure isotherms in relation to hygroscopic water of soils

Six calcareous and alluvial soil profiles differing in their texture, CaCO₃ and salinity were chosen from west and middle Nile Delta for the present study. The 1^st and 2^nd profiles from Borg El-Arab area were sandy loam in texture and > 30% CaCO₃, while the 3^rd and 4^th profiles (from Nubaria area) were sandy clay loam and < 30% CaCO₃. The 2^nd and 4^th profiles were taken from cultivated area with maize. The 5^th profile from Epshan area was non-saline clay alluvial soil and the 6^th from El-Khamsen was saline clay alluvial soil. The relation between soil moisture content (W%) and water vapour pressure (P/P _o) was established for the mentioned soils. Data showed that the specific surface area (S) values were 34–53 and 44–60 m²/g for calcareous soils of Borg El-Arab and Nubaria areas, 206–219 and 206–249 m²/g for non-saline and saline clay alluvial soils of Epshan and El-Khamsen areas, respectively. The corresponding values of the external specific surface area (S _e) were 16–21, 14–22, 72–86 and 92–112 m²/g. Submitting W _m+W _me as an adsorption boundary of moisture films (W _c) (where W _m is mono-adsorbed layer of water vapour on soil particles and W _me is the external mono-adsorbed layer), the maximum water adsorption capacity (W _a) was found to be W _c + W _me or W _m + 2W _me. It was ranged from 1.88 to 2.70%, 1.97 to 2.95%, 9.70–10.70% and 10.80 to 13.12% while the maximum hygroscopic water (M _H) values were 2.43–3.78%, 2.91–4.65%, 16–17% and 18.30–21.9% for the studied soil profiles respectively. The residual moisture content (θ _r) at pF 7 and P/P _o = 0 was ranged from 0.0005–0.0010%, 0.0007–0.0019% and 0.0043–0.0048% in Borg El-Arab, Nubaria and Epshan soil profiles, respectively. The inter-relations between the surface area and the hygroscopic moisture parameters of the soils under investigation were as follows Calcareous soils; W _m = 0.40 M _H, W _c = 0.55 M _H, W _a = 0.70 M _H, S = 14 M _H Non-saline soil; W _m = 0.35 M _H, W _c = 0.49 M _H, W _a = 0.63 M _H, S = 13 M _H Saline soil; W _m = 031 M _H, W _c = 0.45 M _H, W _a = 0.59 M _H, S = 12 M _H These relations give possibility to deduce the soil moisture adsorption capacities and specific surface area via maximum hygroscopic water, which can be obtained through the experimental determination of water vapor adsorption isotherms.     

Perfused transcapillary smooth muscle and endothelial cell co-culture—a novelin vitro model

Summary As mostin vitro endothelial cell (EC)-vascular smooth muscle cell (SMC) co-culture studies have been performed utilizing static culture conditions, none have successfully mimicked the physical environment of these cellsin vivo. EC covering the inner surface of blood vessels are continuously exposed to a hemodynamically imposed mechanical stress resulting from the flow of blood, while SMC are affected by pressure, a flow-related force acting perpendicular to the surface. We have developed a perfused transcapillary co-culture system that permits the chronic exposure of EC and SMC to physiological shear stresses and pressures. SMC and EC co-cultures were successfully established and maintained in long-term culture (7 wk) on an enclosed perfused bundle of semipermeable polypropylene capillaries. By altering flow rate and/or viscosity, shear stresses of 0.07–20 dyn/cm² can be readily achieved in this system. Electron microscopic analysis revealed that SMC formed multilayers around the outside of the capillaries, whereas EC, subjected to 3 dyn/cm² shear stress, formed an intact closely adherent monolayer lining the capillary lumen. EC and SMC exhibited characteristic ultrastructural and gross morphology. EC were separated from SMC by the capillary wall (pore size 0.5 μm, width 150 μM) and while no direct cell-cell contact was evident some cells were seen to migrate into the capillary wall. Both EC and SMC are exposed to the same culture medium, allowing the interaction of substances released in both directions. Yet separate populations of cells are maintained and can be individually harvested for further analysis. This co-culture system that mimics the architecture and physical environment of the vessel wall should have many potential applications in vascular biology.     

Hyaluronic acid depolymerization by ascorbate-redox effects on solid state cultivation of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis> in cashew apple fruit bagasse

The cashew fruit (Anacardium occidentale L.) has been used as a promising agricultural resource for the production of low-molecular weight (M_W) hyaluronic acid (HA) (10⁴–10⁵ Da). The cashew juice is a rich source of vitamin C containing, 1.2–2.0 g L⁻¹. This work explores the effects of the initial concentration of the ascorbate on the solid fermentation of the juice-moisturized bagasse from the cashew apple fruit. The results show that the M_W reduction of HA is proportional to the initial ascorbate concentration. The presence of ascorbate did not influence the Streptococcus zooepidemicus metabolism. However, the HA productivity was increased from 0.18 to 0.28 mg g⁻¹ h⁻¹ when the ascorbate concentration ranged from 1.7 to 10 mg mL⁻¹. These findings contribute to the controlled production of HA in a low M_W range, which is important in cell signalization, angiogenesis and nanoparticles production.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Differential calcium signaling in dairy cows with specific CXCR1 genotypes potentially related to interleukin-8 receptor functionality

Neutrophil migration and activation are critical components of innate immunity and are mediated by a variety of inflammatory mediators, which include interleukin-8 (IL-8) and epithelial-derived neutrophil activating peptide-78 (ENA-78). Limited knowledge on the expression of receptors for these inflammatory mediators (CXCR1 and CXCR2) in bovine, in addition to the association of a polymorphism (G→C) in position +777 of the CXCR1 gene with impaired neutrophil function, prompted evaluation of CXCR1 and CXCR2 mRNA and protein expression, ligand binding affinity, and intracellular receptor signaling in neutrophils from cows with different CXCR1 genotypes. Initial observations revealed that overall IL-8 receptor numbers appeared to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of SB225002, a CXCR2 inhibitor, CXCR1 affinity was about fivefold lower in cows with a CC genotype and may have resulted in an underestimation of receptor numbers in cows with this genotype. In addition, intracellular calcium ([Ca⁺⁺]_i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not ENA-78. Furthermore, when neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, [Ca⁺⁺]_i release was lower in cows with a CC genotype, suggesting differential CXCR1 signaling among genotypes. These findings offer knowledge of the role that each of these receptors plays in the inflammatory response in the bovine and provide insight into the potential mechanisms that may be affected in neutrophils of cows with different CXCR1 genotypes.     

Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces

Endothelial cell (EC) migration plays a critical role in vascular remodeling. Here we investigated the interactions between haptotaxis (induced by extracellular matrix gradient) and mechanotaxis (induced by mechanical forces) during EC migration. A micropatterning technique was used to generate step changes of collagen surface density. Due to haptotaxis, ECs developed focal adhesions and migrated into the area with higher surface density of collagen. Different levels of fluid shear stress were applied on ECs in the direction perpendicular to collagen strips. Shear stress at 2 dyn/cm2 did not affect haptotaxis, while shear stress at 3 dyn/cm2 or higher was sufficient to drive the migration of most ECs in the flow direction and against haptotaxis. Immunostaining revealed the increase of focal adhesions and lamellipodial protrusion in the direction of flow. These results suggest that shear stress beyond a certain threshold can be a predominant factor to determine the direction of EC migration.     

Low-frequency resonance Raman studies of the H(M202)G cavity mutant of bacterial photosynthetic reaction centers

Low-frequency (90–435 cm⁻¹) NIR-excitation (875–900 nm) resonance Raman (RR) studies are reported for the H(M202)G cavity mutant of bacterial photosynthetic reaction centers (RCs) from Rb. sphaeroides that was first described by Goldsmith et al. [(1996) Biochemistry 35: 2421–2428]. In this mutant, the His residue that axially ligates the Mg ion of the M-side bacteriochlorophyll (BChl) of the special pair primary donor (P) is replaced by a non-ligating Gly residue. Regardless, the Mg ion of P_M in the H(M202)G RCs remains pentacoordinates and is presumably ligated by a water molecule, although this axial ligand has not been definitively identified. The low-frequency RR studies of the H(M202)G RCs are accompanied by studies of RCs exchanged with D₂O and incubated with imidazole (Im). The RR studies of the cavity mutant RCs reveal the following: (1) The structure of P_M in the H(M202)G RCs is different from that of the wild-type, consistent with an altered BChl core. (2) A water ligand for P_M in the H(M202)G RCs is generally consistent with the low-frequency RR spectra. The Mg-OH₂ stretching vibration is tentatively assigned to a band at 318 cm⁻¹, a frequency higher than that of the Mg-His stretch of the native pigment (∼ ∼235 cm⁻¹). (3) The BChl core structure of P_M in the cavity mutant is rendered similar (but not identical) to that of the wild-type when the adventitious water axial ligand is replaced by Im. (4) Exchange with D₂O results in more global structural changes, likely involving the protein, which in turn affect the structure of the BChls in P. (5) Assignment of the low-frequency vibrational spectrum of P is generally more complex than originally suggested.     

Metabolic rates in an anadromous clupeid, the American shad (Alosa sapidissima)

To assess the energetics of migration in an anadromous fish, adult American shad (Alosa sapidissima) were swum in a large respirometer at a range of speeds (1.0–2.3 body lengths (BL) s⁻¹, 13–24 °C). Metabolic rate (M_O2) was logarithmically related to swimming speed (Bl s⁻¹; r ² = 0.41, slope = 0.23 ± 0.037) and tailbeat frequency (beats × min⁻¹; r ² = 0.52, slope = 0.003 ± 0.0003). Temperature had a significant effect on metabolic rate (r ² = 0.41) with a Q₁₀ of 2.2. Standard metabolic rate (SMR), determined directly after immobilization with the neuroblocker gallamine triethiodide, ranged from 2.2–6.2 mmolO₂ kg⁻¹ h⁻¹ and scaled with mass (W) such that SMR = 4.0 (±0.03)W^{0.695(±0.15)}. Comparison of directly determined and extrapolated SMR suggests that swimming respirometry provides a good estimate of SMR in this species, given the differences in basal activity monitored by the two methods. Overall, American shad metabolic rates (M_O2 and SMR) were intermediate between salmonids and fast-swimming perciforms, including tunas, and may be a result of evolutionary adaptation to their active pelagic, schooling life history. This study demonstrates variability in metabolic strategy among anadromous fishes that may be important to understanding the relative success of different migratory species under varying environmental conditions. Accepted: 3 March 1999     

A Computational Analysis of Localized Ca<Superscript>2+</Superscript>-Dynamics Generated by Heterogeneous Release Sites

We investigate the role of heterogeneous expression of IP₃R and RyR in generating diverse elementary Ca²⁺ signals. It has been shown empirically (Wojcikiewicz and Luo in Mol. Pharmacol. 53(4):656–662, 1998; Newton et al. in J. Biol. Chem. 269(46):28613–28619, 1994; Smedt et al. in Biochem. J. 322(Pt. 2):575–583, 1997) that tissues express various proportions of IP₃ and RyR isoforms and this expression is dynamically regulated (Parrington et al. in Dev. Biol. 203(2):451–461, 1998; Fissore et al. in Biol. Reprod. 60(1):49–57, 1999; Tovey et al. in J. Cell Sci. 114(Pt. 22):3979–3989, 2001). Although many previous theoretical studies have investigated the dynamics of localized calcium release sites (Swillens et al. in Proc. Natl. Acad. Sci. U.S.A. 96(24):13750–13755, 1999; Shuai and Jung in Proc. Natl. Acad. Sci. U.S.A. 100(2):506–510, 2003a; Shuai and Jung in Phys. Rev. E, Stat. Nonlinear Soft Matter Phys. 67(3 Pt. 1):031905, 2003b; Thul and Falcke in Biophys. J. 86(5):2660–2673, 2004; DeRemigio and Smith in Cell Calcium 38(2):73–86, 2005; Nguyen et al. in Bull. Math. Biol. 67(3):393–432, 2005), so far all such studies focused on release sites consisting of identical channel types. We have extended an existing mathematical model (Nguyen et al. in Bull. Math. Biol. 67(3):393–432, 2005) to release sites with two (or more) receptor types, each with its distinct channel kinetics. Mathematically, the release site is represented by a transition probability matrix for a collection of nonidentical stochastically gating channels coupled through a shared Ca²⁺ domain. We demonstrate that under certain conditions a previously defined mean-field approximation of the coupling strength does not accurately reproduce the release site dynamics. We develop a novel approximation and establish that its performance in these instances is superior. We use this mathematical framework to study the effect of heterogeneity in the Ca²⁺-regulation of two colocalized channel types on the release site dynamics. We consider release sites consisting of channels with both Ca²⁺-activation and inactivation (“four-state channels”) and channels with Ca²⁺-activation only (“two-state channels”) and show that for the appropriate parameter values, synchronous channel openings within a release site with any proportion of two-state to four-state channels are possible, however, the larger the proportion of two-state channels, the more sensitive the dynamics are to the exact spatial positioning of the channels and the distance between channels. Specifically, the clustering of even a small number of two-state channels interferes with puff/spark termination and increases puff durations or leads to a tonic response.     

Endothelium oriented differentiation of bone marrow mesenchymal stem cells under chemical and mechanical stimulations

Bone marrow mesenchymal stem cells (MSCs) have multi-differentiation capability. Their endothelial cell (EC) oriented differentiation is the key to vasculogenesis, in which both mechanical and chemical stimulations play important roles. Most previous studies reported individual effects of VEGF or fluid shear stress (SS), when MSCs were subjected to shear stress of 10–15 dyn/cm² over 24 hr. In this paper, we investigated responses of MSCs from young Sprague Dawley rats to shear stress, VEGF and the combination of the two stimuli. Our study showed that the combined stimulation of shear stress and VEGF resulted in more profound EC oriented differentiation of MSCs in comparison to any individual stimulation. Furthermore, we subjected MSCs to prolonged period of fluid shear stimulation, i.e. 48 hr rather than 24 hr, and increased the magnitude of the shear stress from 10 dyn/cm² to 15, 20 and 25 dyn/cm². We found that without VEGF, the endothelium oriented differentiation of MSCs that was seen following 24 hr of shear stimulation was largely abolished if we extended the shear stimulation to 48 hr. A similar sharp decrease in MSC differentiation was also observed when the magnitude of the shear stress was increased from 10–15 dyn/cm² to 20–25 dyn/cm² in 24 hr shear stimulation studies. However, with combined VEGF and fluid shear stimulation, most of the endothelial differentiation was retained following an extended period, i.e. at 48 hr, of shear stimulation. Our study demonstrates that chemical and mechanical stimulations work together in determining MSC differentiation dynamics.     

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl₂, 0.92 mM NaH₂PO₄ and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS^–8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^–1 thiamine HCl, 100 mg l^–1 myo-inositol, 3.1 g l^–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×10⁵ ml^–1 in 0.4 M MS^–8P for 2–3 weeks, followed by subculture in 0.15 M MS^–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed. Received: 9 February 1998 / Revision received: 4 May 1998 / Accepted: 15 May 1998     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

100% complete assignment of non-labile 1H, 13C, and 15N signals for calcium-loaded calbindin D9k P43G

Here we present the 100% complete assignment chemical shift of non-labile ¹H, ¹⁵N and ¹³C nuclei of Calbindin D_9k P43G. The assignment includes all non-exchangeable side chain nuclei, including ones that are rarely reported, such as LysNζ as well as the termini. NMR experiments required to achieve truly complete assignments are discussed. To the best of our knowledge our assignments for Calbindin D_9k extend beyond previous studies reaching near-completeness (Vis et al. in Biochem 33:14858–14870, 1994; Yamazaki et al. in J Am Chem Soc 116:6464–6465, 1994; Yamazaki et al. in Biochem 32:5656–5669, 1993b).     

Vascular endothelial wound closure under shear stress: role of membrane fluidity and flow-sensitive ion channels.

Sufficiently rapid healing of vascular endothelium following injury is essential for preventing further pathological complications. Recent work suggests that fluid dynamic shear stress regulates endothelial cell (EC) wound closure. Changes in membrane fluidity and activation of flow-sensitive ion channels are among the most rapid endothelial responses to flow and are thought to play an important role in EC responsiveness to shear stress. The goal of the present study was to probe the role of these responses in bovine aortic EC (BAEC) wound closure under shear stress. BAEC monolayers were mechanically wounded and subsequently subjected to either "high" (19 dyn/cm(2)) or "low" (3 dyn/cm(2)) levels of steady shear stress. Image analysis was used to quantify cell migration and spreading under both flow and static control conditions. Our results demonstrate that, under static conditions, BAECs along both wound edges migrate at similar velocities to cover the wounded area. Low shear stress leads to significantly lower BAEC migration velocities, whereas high shear stress results in cells along the upstream edge of the wound migrating significantly more rapidly than those downstream. The data also show that reducing BAEC membrane fluidity by enriching the cell membrane with exogenous cholesterol significantly slows down both cell spreading and migration under flow and hence retards wound closure. Blocking flow-sensitive K and Cl channels reduces cell spreading under flow but has no impact on cell migration. These findings provide evidence that membrane fluidity and flow-sensitive ion channels play distinct roles in regulating EC wound closure under flow.     

Purification of the photosynthetic reaction center from <Emphasis Type="Italic">Heliobacterium modesticaldum</Emphasis>

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006; Romberger et al., Photosynth Res 104:293–303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c ₅₅₃ and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g′, two 8¹-OH-Chl a _F, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the F_A and F_B [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P₈₀₀ ⁺F_X⁻ state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a K _M of 10 μM and a k _cat of 9.5 s⁻¹ under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.     

Short-Term Shear Stress Induces Rapid Actin Dynamics in Living Endothelial Cells

Hemodynamic shear stress guides a variety of endothelial phenotype characteristics, including cell morphology, cytoskeletal structure, and gene expression profile. The sensing and processing of extracellular fluid forces may be mediated by mechanotransmission through the actin cytoskeleton network to intracellular locations of signal initiation. In this study, we identify rapid actin-mediated morphological changes in living subconfluent and confluent bovine aortic endothelial cells (ECs) in response to onset of unidirectional steady fluid shear stress (15 dyn/cm²). After flow onset, subconfluent cells exhibited dynamic edge activity in lamellipodia and small ruffles in the downstream and side directions for the first 12 min; activity was minimal in the upstream direction. After 12 min, peripheral edge extension subsided. Confluent cell monolayers that were exposed to shear stress exhibited only subtle increases in edge fluctuations after flow onset. Addition of cytochalasin D to disrupt actin polymerization served to suppress the magnitude of flow-mediated actin remodeling in both subconfluent confluent EC monolayers. Interestingly, when subconfluent ECs were exposed to two sequential flow step increases (1 dyn/cm² followed by 15 dyn/cm² 12 min later), actin-mediated edge activity was not additionally increased after the second flow step. Thus, repeated flow increases served to desensitize mechanosensitive structural dynamics in the actin cytoskeleton.     

Kinetic models of photosystem II should accommodate the effect of donor side quenching on variable chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence in the microseconds time range

Quantitative data on laser flash-induced variable fluorescence in the 100 ns to 1 ms time range (Belyaeva et al. in Photosynth Res 98:105–119, 2008) confirming those of others (Steffen et al. in Biochemistry 40:173–180, 2001, Biochemistry 44:3123–3132, 2005; Belyaeva et al. in Biophysics 51(6):976–990, 2006), need a substantial correction with respect to magnitude of the normalized variable fluorescence associated with single turnover-induced charge separation in RCs of PS II. Their data are conclusive with the involvement of donor side quenching, the release of which occurs with a rate constant in the range of tens of ms⁻¹, and presumed to be associated with reduction of Y_\textz ⁺ Y_{\text{z}}^{ + } by the OEC.