Energy-dependent Fashion Research Articles

Laser Biostimulation Induces Wound Healing-Promoter β2-Defensin Expression in Human Keratinocytes via Oxidative Stress.

The innate immune system is the first line of defense of the body composed of anatomical barriers, such as skin and mucosa, as well as effector cells, antimicrobial peptides, soluble mediators, and cell receptors able to detect and destroy viruses and bacteria and to sense trauma and wounds to initiate repair. The human β-defensins belong to a family of antimicrobial small cationic peptides produced by epithelial cells, and show immunomodulatory and pro-healing activities. Laser biostimulation is a therapy widely used to contrast microbial infection and to accelerate wound healing through biological mechanisms that include the creation of oxidative stress. In this paper, we explored laser biostimulation's ability to modulate the production of two β-defensins, hBD-1 and hBD-2, in human keratinocytes and whether this modulation was, at least in part, oxidative-stress-dependent. Human spontaneously immortalized keratinocytes (HaCaT) were stimulated using laser irradiation at a 980 nm wavelength, setting the power output to 1 W (649.35 mW/cm2) in the continuous mode. Cells were irradiated for 0 (negative control), 5, 10, 25 and 50 s, corresponding to an energy stimulation of 0, 5, 10, 25 and 50 J. Positive control cells were treated with lipopolysaccharide (LPS, 200 ng/mL). After 6 and 24 h of treatment, the cell conditioned medium was collected and analyzed via ELISA assay for the production of hBD-1 and hBD-2. In another set of experiments, HaCaT were pre-incubated for 45 min with antioxidant drugs-vitamin C (Vit. C, 100 µM), sodium azide (NaN3, 1 mM); ω-nitro-L-arginine methyl ester (L-NAME, 10 mM) and sodium pyruvate (NaPyr, 100 µM)-and then biostimulated for 0 or 50 s. After 6 h, the conditioned medium was collected and used for the ELISA analysis. The hBD-1 and hBD-2 production by HaCaT was significantly increased by single laser biostimulation after 6 h in an energy-dependent fashion compared to basal levels, and both reached production levels induced by LPS. After 24 h, only hBD-2 production induced by laser biostimulation was further increased, while the basal and stimulated hBD-1 levels were comparable. Pre-incubation with antioxidative drugs was able to completely abrogate the laser-induced production of both hBD-1 and hBD-2 after 6 h, with the exception of hBD-1 production in samples stimulated after NaN3 pre-incubation. A single laser biostimulation induced the oxidative-stress-dependent production of both hBD-1 and hBD-2 in human keratinocytes. In particular, the pro-healing hBD-2 level was almost three times higher than the baseline level and lasted for 24 h. These findings increase our knowledge about the positive effects of laser biostimulation on wound healing.

Termination of the Activating NK Cell Immunological Synapse Is an Active and Regulated Process.

Cellular cytotoxicity is essential for the elimination of virus-infected and cancerous cells by NK cells. It requires a direct cellular contact through the establishment of an immunological synapse (IS) between the NK cell and the target cell. In this article, we show that not only the establishment of the IS, but also its maintenance is a highly regulated process. Ongoing receptor-proximal signaling events from activating NK cell receptors and actin dynamics were necessary to maintain a stable contact in an energy-dependent fashion, even after the IS was formed successfully. More importantly, the initiation of a contact to a new susceptible target cell resulted in accelerated detachment from an old target cell. We propose that the maintenance of an existing IS is a dynamic and regulated process to allow for effective serial killing of NK cells.

Genome-Wide Comparative In Silico Analysis of the RNA Helicase Gene Family in Zea mays and Glycine max: A Comparison with Arabidopsis and Oryza sativa

RNA helicases are enzymes that are thought to unwind double-stranded RNA molecules in an energy-dependent fashion through the hydrolysis of NTP. RNA helicases are associated with all processes involving RNA molecules, including nuclear transcription, editing, splicing, ribosome biogenesis, RNA export, and organelle gene expression. The involvement of RNA helicase in response to stress and in plant growth and development has been reported previously. While their importance in Arabidopsis and Oryza sativa has been partially studied, the function of RNA helicase proteins is poorly understood in Zea mays and Glycine max. In this study, we identified a total of RNA helicase genes in Arabidopsis and other crop species genome by genome-wide comparative in silico analysis. We classified the RNA helicase genes into three subfamilies according to the structural features of the motif II region, such as DEAD-box, DEAH-box and DExD/H-box, and different species showed different patterns of alternative splicing. Secondly, chromosome location analysis showed that the RNA helicase protein genes were distributed across all chromosomes with different densities in the four species. Thirdly, phylogenetic tree analyses identified the relevant homologs of DEAD-box, DEAH-box and DExD/H-box RNA helicase proteins in each of the four species. Fourthly, microarray expression data showed that many of these predicted RNA helicase genes were expressed in different developmental stages and different tissues under normal growth conditions. Finally, real-time quantitative PCR analysis showed that the expression levels of 10 genes in Arabidopsis and 13 genes in Zea mays were in close agreement with the microarray expression data. To our knowledge, this is the first report of a comparative genome-wide analysis of the RNA helicase gene family in Arabidopsis, Oryza sativa, Zea mays and Glycine max. This study provides valuable information for understanding the classification and putative functions of the RNA helicase gene family in crop growth and development.

Dynamically optimized Wang-Landau sampling with adaptive trial moves and modification factors

The density of states of continuous models is known to span many orders of magnitudes at different energies due to the small volume of phase space near the ground state. Consequently, the traditional Wang-Landau sampling which uses the same trial move for all energies faces difficulties sampling the low-entropic states. We developed an adaptive variant of the Wang-Landau algorithm that very effectively samples the density of states of continuous models across the entire energy range. By extending the acceptance ratio method of Bouzida, Kumar, and Swendsen such that the step size of the trial move and acceptance rate are adapted in an energy-dependent fashion, the random walker efficiently adapts its sampling according to the local phase space structure. The Wang-Landau modification factor is also made energy dependent in accordance with the step size, enhancing the accumulation of the density of states. Numerical simulations show that our proposed method performs much better than the traditional Wang-Landau sampling.

Ghrelin and bone

Ghrelin is a gut-derived peptide hormone, first isolated from the stomach. Ghrelin was initially characterized as a growth hormone (GH) secretagogue, but it plays a more important role as a potent orexigen and modulator of whole-body energy homeostasis. Ghrelin itself is closely regulated by metabolic status. Bone remodeling constantly renews the skeleton in a highly energy-dependent fashion. Accordingly, bone metabolism is tightly coupled to energy metabolism through the integration of peripheral and central mechanisms, involving the sympathetic nervous system and factors such as leptin. Ghrelin has been shown to modulate osteoblast differentiation and function, both directly and perhaps also through regulation of the GH-insulin-like growth factor axis. However, recently it has also been shown that ghrelin interacts with leptin in modulating bone structure, constituting a new mechanism that couples bone metabolism with energy homeostasis. In this review, we discuss the role that ghrelin plays modulating bone cell function, and its integrative role in coupling bone metabolism with energy metabolism.

Purified sakacin A shows a dual mechanism of action against Listeria spp: proton motive force dissipation and cell wall breakdown

Sakacin A was purified to homogeneity through simple chromatographic procedures from cultures of Lactobacillus sakei DSMZ 6333 grown on a low-cost medium. The highly purified protein dissipated both transmembrane potential (ΔΨ) and transmembrane pH gradient (ΔpH) in Listeria cells in a very intense, rapid, and energy-dependent fashion. On a slower timescale, purified sakacin A also showed a lytic activity toward isolated cell walls of Listeria. Mass spectrometry was used to analyze the products of sakacin A action on cell walls, evidencing that sakacin A acts on various types of bonds within peptoglycans.

Characterization of reduced and oxidized dopamine and 3,4-dihydrophenylacetic acid, on brain mitochondrial electron transport chain activities

Loss of dopamine (DA) homeostasis may be a contributing factor to cell damage in Parkinson's disease (PD). Past studies showing deleterious effects of DA on mitochondrial function, however, have been inconsistent raising questions about mitochondria as a downstream target for DA. Issues such as the dopamine species i.e., reduced or oxidized, time of exposure and the effect of major metabolites such as 3,4-dihydrophenylacetic acid (DOPAC) may contribute to the disparate findings. The present study used isolated, lysed rat brain mitochondria to characterize the effects of oxidized or reduced DA and DOPAC on complex activities of the electron transport chain (ETC). Time of exposure and quantitation of reduced or oxidized catachols for DA and DOPAC were monitored for all experiments. Reduced DA and DOPAC with or without a 30 min preincubation had no affect on NADH oxidase activity which monitors the activities of complexes I, III and IV. Complex II activity was inhibited by reduced DA (≥ 500 μM), but not by reduced DOPAC and was significantly attenuated by SOD suggesting reactive oxygen species involvement. In contrast, fully oxidized DA and DOPAC dose dependently inhibited NADH oxidase, complex I and complex III activities with IC 50s in the 50–200 μM range. No preincubation was required for inhibition with the catechols when they were fully oxidized. Oxidized DA inhibited complex I only when exposure occurred during stimulated electron flow, suggesting covalent binding of quinones to proteins within active sites of the complex. In intact, well coupled mitochondria, extramitochondrial DA was shown to access the mitochondrial matrix in a dose, time and energy-dependent fashion. The findings suggest that many of the reported inconsistencies with regards to the effects of DA and DOPAC on ETC function can be attributed to the oxidized state of the catechol at the time of exposure. In addition, the findings provide possible downstream targets for DA that could contribute to the vulnerability of dopaminergic neurons in PD.

Nuclear Import of HSV-1 DNA Polymerase Processivity Factor UL42 Is Mediated by a C-Terminally Located Bipartite Nuclear Localization Signal

The polymerase accessory protein of the human herpes simplex virus type 1 (HSV-1) DNA polymerase UL42 plays an essential role in viral replication, conferring processivity to the catalytic subunit UL30. We show here that UL42 is imported to the nucleus of living cells in a Ran- and energy-dependent fashion, through a process that requires a C-terminally located bipartite nuclear localization signal (UL42-NLSbip; PTTKRGRSGGEDARADALKKPK(413)). Moreover cytoplasmic mutant derivatives of UL42 lacking UL42-NLSbip are partially relocalized into the cell nucleus upon HSV-1 infection or coexpression with UL30, implying that the HSV-1 DNA polymerase holoenzyme can assemble in the cytoplasm before nuclear translocation occurs, thus explaining why the UL42 C-terminal domain is not strictly required for viral replication in cultured cells. However, mutation of both UL30 and UL42 NLS results in retention of the DNA polymerase holoenzyme in the cytoplasm, suggesting that simultaneous inhibition of both NLSs could represent a viable strategy to hinder HSV-1 replication. Intriguingly, UL42-NLSbip is composed of two stretches of basic amino acids matching the consensus for classical monopartite NLSs (NLSA, PTTKRGR(397); NLSB, KKPK(413)), neither of which are capable of targeting GFP to the nucleus on their own, consistent with the hypothesis that P and G residues in position +3 of monopartite NLSs are not compatible with nuclear transport in the absence of additional basic sequences located in close proximity. Our results showing that substitution of G or P of the NLS with an A residue partially confers NLS function will help to redefine the consensus for monopartite NLSs.

Co-localization of galectin-1 with GM1 ganglioside in the course of its clathrin- and raft-dependent endocytosis.

Mammalian galectin-1 (Gal-1), a beta-galactoside-binding lectin has a prominent role in regulating cell adhesion, cell growth and immune responses. Downregulation of these biological functions may occur via internalization of Gal-1. In the present study we have investigated the mechanism and possible mediator(s) of Gal-1 endocytosis. We show that internalization occurs at a temperature higher than 22 degrees C in an energy dependent fashion. After one hour incubation Gal-1 localizes in the Golgi system within the cells, and then disappears without accumulation in degradation compartments, such as lysosomes. Based on their strong intracellular co-localization, two glycoconjugates, GM1 ganglioside and CD7 are implicated in the sorting of internalized Gal-1 into Golgi. Other known Gal-1 binding glycoproteins on T cells (CD2, CD3, CD43 and CD45) do not cointernalize with the lectin. Internalization of Gal-1 depends on its lectin activity and follows dual pathways involving clathrin-coated vesicles and raft-dependent endocytosis.

A Novel Ded1-like RNA Helicase Interacts with the Y-box Protein ctYB-1 in Nuclear mRNP Particles and in Polysomes

We have characterized a novel mRNA-binding protein, designated hrp84, in the dipteran Chironomus tentans and identified it as a DEAD-box RNA helicase. The protein contains the typical helicase core domain, a glycine-rich C-terminal part and a putative nuclear export signal in the N terminus. The protein belongs to the Ded1 subgroup of DEAD-box helicases, which is highly conserved from yeast (Ded1p) to mammals (DDX3). In tissue culture cells, hrp84 is present both in the nucleus and cytoplasm and, as shown by in vivo UV cross-linking, is bound to mRNA in both compartments. Immunoprecipitation experiments revealed that hpr84 is associated with the C. tentans homologue (ctYB-1) of the vertebrate Y-box protein YB-1 both in the nucleus and cytoplasm, and the two proteins also appear together in polysomes. The interaction is likely to be direct as shown by in vitro binding of purified components. We conclude that the mRNA-bound hrp84.ctYB-1 complex is formed in the nucleus and is translocated with mRNA into the cytoplasm and further into polysomes. As both Ded1 and YB-1 are known to regulate the initiation of translation, we propose that the RNA helicase-Y-box protein complex affects the efficiency of mRNA translation, presumably by modulating the conformation of the mRNP template.

Mechanism of Uptake of C105Y, a Novel Cell-penetrating Peptide

C105Y, a synthetic peptide (CSIPPEVKFNKPFVYLI) based on the amino acid sequence corresponding to residues 359-374 of alpha1-antitrypsin, enhances gene expression from DNA nanoparticles. To investigate how this enhancement occurs, C105Y was fluorescently labeled to study its uptake and intracellular trafficking. When human hepatoma cells (HuH7) were incubated with fluorescently labeled C105Y for as little as 3 min, C105Y displayed nuclear and cytoplasmic staining with enrichment of fluorescent signal in the nucleus and nucleolus. Uptake and nucleolar localization were observed with the short sequence PFVYLI, but not with SIPPEVKFNK, and the D-isomer was readily taken up into cells but not into the nucleus. We found that the C105Y peptide is routed to the nucleolus very rapidly in an energy-dependent fashion, whereas membrane translocation and nuclear localization are energy-independent. When we tested the involvement of known endocytosis pathways in uptake and trafficking of this peptide, we demonstrated that C105Y peptide is internalized by a clathrin- and caveolin-independent pathway, although lipid raft-mediated endocytosis may play a role in peptide intracellular trafficking. Efficient energy-independent cell entry with rapid nuclear localization probably accounts for enhancement of gene expression from inclusion of C105Y into DNA nanoparticles.

RNA Expression Levels of (Co)Chaperone Molecules of the Glucocorticoid Receptor Are Not Related to In Vitro Prednisolone Resistance in Childhood ALL.

In vitro resistance to Glucocorticoids (GC) is an important adverse risk factor in the treatment of ALL. To induce apoptosis in ALL cells, GC have to bind to the GC receptor (hGR), which is tightly regulated by various (co)chaperone molecules. HSP70, ST13 and HSP40 facilitate hGR binding to HSP90 and HOP in an energy dependent fashion. This complex is stabilised by immunophillins FKBP 51, FKBP59, CYP40 and P23 and is necessary for the hGR to be able to bind GC. The ATPase BAG1 can function as negative regulator of HSP70, and may downregulate hGR activity. After the GC-hGR complex is formed, it is transported to the nucleus to regulate GC responsive genes. In this study, we tested the hypothesis that RNA expression levels of these (co) chaperone molecules are important determinants for in vitro prednisolone sensitivity in childhood ALL.Methods: 20 children with leukemic cells in vitro sensitive to prednisolone (LC50<0.1μg/ml) were matched (according to age, immunophenotype and white blood cell count at diagnosis) each with an in vitro resistant patient (LC50 > 150 μg/ml). RNA expression levels of the different (co)chaperone molecules were measured by a quantitative real-time RT-PCR (Taqman) assay and standardised to endogenous GAPDH and RNAseP mRNA levels. In vitro resistance to prednisolone was measured by the MTT assay.Results. The highest median expression levels, indicated as percentage of GAPDH levels were found for HSP90 (29%) and p23 (10%), whereas FKBP51 and ST13 were expressed at 2% and 1.6% respectively. HSP70 (0.1%), HSP40 (0.44%), HOP (0.2%), FKBP59 (0.4%), PPID (0.3%) and BAG-1 (0.4%) were expressed at relatively low levels. Using matched pair analysis, no significant differences were found for the expression levels of the various (co)chaperone molecules between in vitro sensitive and resistant patients. The ratio of different (co)chaperone molecules with opposing effect (HIP versus BAG-1, FKBP-51 versus FKBP-52, HSP-90 versus P23, and HSP-90 versus the functional GR-alpha receptor, positive and negative effect respectively) was not related to GC resistance as well.Conclusion: Glucocorticoid resistance in childhood ALL can not be attributed to basal expression levels of the diverse (co)chaperone molecules involved in GC binding and transport of the GC-GR complex to the nucleus of the GR.

Identification of cryptic nuclear localization signals in the prion protein

Abnormal transport of C-terminally truncated prion protein (PrP) to the nucleus has been reported in cell models of familial prion disorders associated with a stop codon mutation at residues 145 or 160 of the PrP. In both cases, PrP is translocated to the nucleus in an energy-dependent fashion, implying the presence of cryptic nuclear localization signal(s) in this region of PrP. In this report, we describe the presence of two independent nuclear localization signals (NLS) in the N-terminal domain of PrP that differ in the efficiency of nuclear targeting. When acting independently, each NLS sequence mediates the transport of tagged bovine serum albumin into the nucleus of permeabilized cells. When acting together, the two NLS sequences complement each other in transporting the N-terminal fragment of PrP to the nucleus of transfected cells, where it accumulates at steady state. Interestingly, nuclear translocation of PrP is blocked completely if the N-terminal fragment is extended to include one or two N-glycans. The glycosylated PrP fragment, instead, accumulates in the endoplasmic reticulum. Extension of the N-terminal fragment to include both N-glycans and the glycosyl phosphatidylinositol anchor, as expected, directs PrP to the plasma membrane. These observations hold implications for the pathogenesis of familial prion disorders, where truncated and abnormally glycosylated mutant PrP forms may accumulate in the nucleus and initiate neurotoxicity through novel mechanisms.

Chromosome dynamics in the yeast interphase nucleus.

Little is known about the dynamics of chromosomes in interphase nuclei. By tagging four chromosomal regions with a green fluorescent protein fusion to lac repressor, we monitored the movement and subnuclear position of specific sites in the yeast genome, sampling at short time intervals. We found that early and late origins of replication are highly mobile in G1 phase, frequently moving at or faster than 0.5 micrometers/10 seconds, in an energy-dependent fashion. The rapid diffusive movement of chromatin detected in G1 becomes constrained in S phase through a mechanism dependent on active DNA replication. In contrast, telomeres and centromeres provide replication-independent constraint on chromatin movement in both G1 and S phases.

Interaction of S100A8/S100A9-arachidonic acid complexes with the scavenger receptor CD36 may facilitate fatty acid uptake by endothelial cells.

Recently, we showed that S100A8/A9 were secreted from phorbol ester-stimulated neutrophil-like HL-60 cells, thereby carrying arachidonic acid [Kerkhoff et al. (1999) J. Biol. Chem. 274, 32672-32679]. The present study was undertaken to evaluate whether the secreted S100A8/A9-AA complex might be involved in transcellular eicosanoid metabolism. In the presence of S100A8/A9, arachidonic acid was rapidly taken up by human umbilical vein endothelial cells in a saturable and energy-dependent fashion. Protein-facilitated arachidonate uptake was confirmed by its sensitivity toward the protein modifiers bromobimane and phloretin. Both potassium and sodium depletion did not affect the arachidonate transport, indicating that arachidonate influx was independent of endocytosis. The uptake of exogenous arachidonic acid by HUVEC was predominantly mediated by FAT/CD36. This conclusion was drawn by the findings that (i) arachidonate uptake was drastically inhibited by sulfo-N-succinimidyl oleate, a specific inhibitor of FAT/CD36; (ii) the maximal inhibition of arachidonate uptake induced by SSO was similar to that effected by ATP depletion; and (iii) the arachidonate transport was 2-fold higher in COS-7 cells transfected with the pEF.BOS-CD36 expression vector than in the empty vector-transfected COS-7 cells. Kinetic studies of arachidonate uptake were indicative for an interaction between fatty acid transporter and S100A8/A9-AA complex that was confirmed by an in vitro protein-protein interaction assay. FAT/CD36 has been suggested to be involved in inflammatory responses, and S100A8/A9 are released from activated leukocytes at inflammatory loci. Therefore, it can be envisioned that their interaction might propagate host response by perpetuating recruitment and activation of cellular effectors.

The Carboxyl Terminus of Interferon-γ Contains a Functional Polybasic Nuclear Localization Sequence

Cytokines such as interferon-gamma (IFN-gamma), which utilize the well studied JAK/STAT pathway for nuclear signal transduction, are themselves translocated to the nucleus. The exact mechanism for the nuclear import of IFN-gamma or the functional role of the nuclear translocation of ligand in signal transduction is unknown. We show in this study that nuclear localization of IFN-gamma is driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of a heterologous protein allophycocyanin (APC) in standard import assays in digitonin-permeabilized cells. Similar to other nuclear import signals, we show that a peptide representing amino acids 95-132 of IFN-gamma (IFN-gamma(95-132)) containing the polybasic sequence 126RKRKRSR132 was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Moreover, deletions immediately NH2-terminal of this sequence did not affect the nuclear import. Thus, the sequence 126RKRKRSR132 is necessary and sufficient for nuclear localization. Furthermore, nuclear import was strongly blocked by competition with the cognate peptide IFN-gamma(95-132) but not the peptide IFN-gamma(95-125), which is deleted in the polybasic sequence, further confirming that the NLS properties were contained in this sequence. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-gamma(95-132). This observation suggests that the NLS in IFN-gamma may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-gamma, when coupled to APC, was also able to mediate its nuclear import. Again, nuclear import was blocked by the peptide IFN-gamma(95-132) and the SV40 T-NLS peptide, suggesting that intact IFN-gamma was also transported into the nucleus through the Ran/importin pathway. Previous studies have suggested a direct intracellular role for IFN-gamma in the induction of its biological activities. Based on our data in this study, we suggest that a key intracellular site of interaction of IFN-gamma is the one with the nuclear transport mechanism that occurs via the NLS in the COOH terminus of IFN-gamma.

Affinity modulation of platelet integrin alphaIIbbeta3 by beta3-endonexin, a selective binding partner of the beta3 integrin cytoplasmic tail.

Platelet agonists increase the affinity state of integrin alphaIIbbeta3, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. beta3-Endonexin is a novel 111-amino acid protein that binds selectively to the beta3 tail. Since beta3-endonexin is present in platelets, we asked whether it can affect alphaIIbbeta3 function. When beta3-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor alphaIIbbeta3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and alphaIIbbeta3 bound little or no PAC1. However, those transfected with GFP/beta3-endonexin and alphaIIbbeta3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/beta3-endonexin did not affect levels of surface expression of alphaIIbbeta3 nor did it modulate the affinity of an alphaIIbbeta3 mutant that is defective in binding to beta3-endonexin. Affinity modulation of alphaIIbbeta3 by GFP/beta3-endonexin was inhibited by coexpression of either a monomeric beta3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that beta3-endonexin can modulate the affinity state of alphaIIbbeta3 in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein-protein interactions at the level of the cytoplasmic tails of alphaIIbbeta3.

The effect of nonablative laser energy on joint capsular properties. An in vitro histologic and biochemical study using a rabbit model.

The purpose of this study was to evaluate the effect of laser energy at nonablative levels on joint capsular histologic and biochemical properties in an in vitro rabbit model. The medial and lateral portions of the femoropatellar joint capsule from both stifles of 12 mature New Zealand White rabbits were used. Specimens were divided into three treatment groups (5 watts, 10 watts, and 15 watt) and one control group using a randomized block design. Specimens were placed in a 37 degrees bath of lactated Ringer's solution and laser energy was applied using a holmium:yttrium-aluminum-garnet laser in four transverse passes across the tissue at a velocity of 2 mm/sec with the handpiece set 1.5 mm from the synovial surface. Histologic analysis revealed thermal alteration of collagen (fusion) and fibroblasts (pyknosis) at all energy densities, with higher laser energy causing significantly greater morphologic changes over a larger area (P < 0.05). Application of laser energy did not significantly alter the biochemical parameters evaluated, including type I collagen content and nonreducible crosslinks (P > 0.05). This study demonstrated that nonablative laser energy caused significant thermal damage to the joint capsular tissue in an energy-dependent fashion, but type I collagen content and nonreducible crosslinks (P > 0.05). This study demonstrated that nonablative laser energy caused significant thermal damage to the joint capsular tissue in an energy-dependent fashion, but type I Collagen content and nonreducible corsslinks were not significantly altered.

Selective inhibition of neutrophil function by a peptide derived from lipocortin 1 N-terminus

A multi-faceted approach was used to investigate the effect of an anti-inflammatory peptide derived from human lipocortin 1 N-terminus region (amino acid 2–26; termed human Ac2–26) on human neutrophil activation in vitro. When incubated with purified human neutrophils. human Ac2–26 produced a concentration-dependent inhibition of elastase release stimulated by formyl-Met-Leu-Phe (fMLP), platelet-activating factor, or leukotriene B 4, with an approximate EC 50 of 33 μM (100 μg/ml). At this concentration, human Ac2–26 also inhibited (77%) the release of [ 3H]-arachidonic acid from neutrophils stimulated with fMLP. The peptide, however, did not inhibit the up-regulation of the β 2-integrin CD11b and the concomitant shedding of L-selectin from neutrophil plasma membrane induced by fMLP. In adhesion experiments, human Ac2–26 inhibited neutrophil adhesion to endothelial monolayers when this was stimulated with fMLP, but not when this followed endothelial cell activation with histamine or platelet-activating factor. Again, the effect of the peptide was concentration-dependent, and an approximate EC 50 of 33 μM was calculated. When a preparation of 125I-labeled human Ac2–26 was incubated with the neutrophils, the peptide was internalised in an energy-dependent fashion. All together, these observations lead us to propose a model in which this peptide derived from the N-terminus of human lipocortin 1 alters a common cellular mechanism producing a selective inhibition of neutrophil activation.

Cytokine-activated endothelial cells internalize E-selectin into a lysosomal compartment of vesiculotubular shape. A tubulin-driven process.

The fate of E-selectin expressed on TNF-activated monolayers of HUVEC was investigated by confocal laser scanning microscopy. Cytokine-activated endothelial cells internalized mAb to E-selectin in a very rapid, energy-dependent fashion. By contrast, mAb against ICAM-1 and VCAM-1 remained surface bound. The E-selectin mAb was recovered in intracellular compartments with a tubular morphology, some of which appeared to be interconnected. Cathepsin B, a ubiquitously expressed lysosomal protease, was found to co-localize in these structures. Functional specificity of E-selectin-internalization was observed upon addition of the fluorescent SLex-oligosaccharide to the activated HUVEC monolayers. Uptake into the same E-selectin-positive compartments was observed, whereas the control oligosaccharide Lex was not internalized at all. The process of internalization was found to be unaffected by most inhibitors of protein kinase C, cAMP-dependent PKA, or protein tyrosine kinase activity. Whereas cytochalasin B preincubation of HUVEC failed to inhibit the internalization process, colchicine and vinblastine, reagents that interfere with the metabolism of tubulin, prevented the formation of the elongated structures in which E-selectin would normally be internalized. Concomitantly, the expression of E-selectin at the cell surface was significantly increased.