Activated G protein α s subunits increase the ethanol sensitivity of human glycine receptors

It is well known that ethanol modulates the function of the Cys loop ligand-gated ion channels, which include the inhibitory glycine receptors (GlyRs). Previous studies have consistently shown that transmembrane and extracellular sites are essential for ethanol actions in GlyRs. In addition, recent evidence has shown that the ethanol modulation of GlyRs is also affected by G protein activation through Gβγ subunits. However, more specific roles of G protein α subunits on ethanol actions are unknown. Here, we show that the allosteric effect of ethanol on the human α(1) GlyR is selectively enhanced by the expression of Gα(s) Q-L. For example, constitutively active Gα(s), but not Gα(q) or Gα(i), was able to displace the alcohol sensitivity of GlyRs toward low millimolar concentrations (17 ± 4 versus 48 ± 5% at 100 mM). Experiments under conditions that increased cAMP and protein kinase A (PKA)-mediated signaling, on the contrary, did not produce the same enhancement in sensitivity, suggesting that the Gα(s) Q-L effect was not dependent on cAMP/PKA-dependent signaling. On the other hand, the effect of Gα(s) Q-L was blocked by a Gβγ scavenger (9 ± 3% of control). Furthermore, two mutant receptors previously shown to have impaired interactions with Gβγ were not affected by Gα(s) Q-L, suggesting that Gβγ is needed for enhancing ethanol sensitivity. These results support the conclusion that activated Gα(s) can facilitate the Gβγ interaction with GlyRs in presence of ethanol, independent of increases in cAMP signaling. Thus, these data indicate that the activated form of Gα(s) is able to positively influence the effect of ethanol on a type of inhibitory receptor important for motor control, pain, and respiration.     

Quantitative analysis of activating alpha subunit of the G protein (Gsα) mutation by pyrosequencing in fibrous dysplasia and other bone lesions

Benign fibro-osseous lesions (BFOLs) frequently display overlapping histological features. The differentiation of fibrous dysplasia (FD) from other BFOLs can be difficult, even for experienced orthopedic pathologists. Accurately distinguishing FD from other BFOLs may have significant clinical and treatment implications. A somatic mutation in gene GNAS encoding the α subunit of the G protein (Gsα) involving the codon corresponding to Arg 201 has been identified in FD and is specifically absent in other BFOLs. We have developed a quantitative assay by pyrosequencing that has a detection sensitivity of 95%. The test allows the identification of the two most common types of mutation (Arg→His and Arg→Cys) in a single reaction, with the ability to analyze other rare mutations. Of the 24 FD cases in this series, 23 (96%) were positive for GNAS/Gsα mutation. Nineteen of 23 positive cases exhibited a G→A mutation (Arg→His), whereas four had a C→T mutation (Arg→Cys). One of three BFOL, not otherwise specified cases was positive for G→A mutation. None of the osteofibrous dysplasia, ossifying fibromas, or other bone lesions were positive for this mutation. Our experience is that pyrosequencing is an easy and accurate quantification method for Gsα mutation detection in fibrous dysplasia. Mutation analysis of the Gsα by pyrosequencing has significant potential for improving discrimination between FD and other BFOLs in problematic cases.     

The treatment of spastic cerebral palsy by side—to—side neurorrhaphy of perpheral nerves

Objective :To discuss a new method and its mechanism for the treatment of spastic cerebral palsy.Methods:6 case were treated.The injurious nerve trunk was kept abreast of neithbor donor nerve at suitable segement,the epineurium and fascicu of two neighboring area were incised to appear nerve fibers,then side-to-side anastomosed each other through the epineurium and fascicu.Result All cases were followed-up for 4-15 months.The spastic limb and deformity of all cases have obvious relieve,5 cases had no spasm without extra stimulation and have the main function recovered,Conclusion Side-to-side neruorrhaphy is a new method to treat spastic cerebral palsy ,After operation,the spatstic muscle could obtain normal never control,thereby,the cervical orientation area was changed.     

The A-B-C of small-molecule ABC transport protein modulators: From inhibition to activation—a case study of multidrug resistance-associated protein 1 (ABCC1)

Several mechanisms of pharmacokinetic, metabolic, and regulatory nature have been elucidated to take part or act in concert in the phenomenon of multidrug resistance (MDR). MDR is characterized by cross-resistance of cells against chemotherapeutic agents, which are used for treatment of e.g., cancer, bacterial infections, or human immunodeficiency virus (HIV) infections. One group of proteins that combines all three stated aspects—the metabolism and distribution of drugs as well as their own regulation—is adenosine triphosphate-binding cassette (ABC) transporters. These efflux pumps use the energy of adenosine triphosphate hydrolysis for drug translocation from the membrane and the cytosol to the extracellular space, often with cotransport of a cosubstrate. Multidrug resistance-associated protein 1 (MRP1, ABCC1) had been discovered as one major key player in cancer-related MDR. The xenobiotic substrates include anthracyclines, vinca alkaloids, podophyllotoxins, as well as glutathione (GSH)-adducts of certain cytostatics. Contrary to other transport proteins involved in cancer-related MDR the activity of MRP1 is related to the GSH content of cells. A modern strategy to overcome MRP1-associated MDR is besides its inhibition the activation of GSH efflux, enforcing cell death due to cellular stress. In addition, it has recently been found that MRP1 contributes to the β-amyloid protein clearance in Alzheimer's disease (AD). Collectively, transport activation of MRP1 is of therapeutic value, and furthermore helps to elucidate the transport protein function and the mechanisms behind it. This review is meant to summarize the known concepts of MRP1 activation, which might contribute to a further understanding of MRP1 in particular and ABC transporters in general.     

Drug delivery to the brain by focused ultrasound induced blood–brain barrier disruption: Quantitative evaluation of enhanced permeability of cerebral vasculature using two-photon microscopy

Reversible and localized blood–brain barrier disruption (BBBD) using focused ultrasound (FUS) in combination with intravascularly administered microbubbles (MBs) has been established as a non-invasive method for drug delivery to the brain. Using two-photon fluorescence microscopy (2PFM), we imaged the cerebral vasculature during BBBD and observed the extravasation of fluorescent dye in real-time in vivo. We measured the enhanced permeability upon BBBD for both 10 kDa and 70 kDa dextran conjugated Texas Red (TR) at the acoustic pressure range of 0.2–0.8 MPa and found that permeability constants of TR10kDa and TR70kDa vary from 0.0006 to 0.0359 min^− 1 and from 0.0003 to 0.0231 min^− 1, respectively. For both substances, a linear regression was applied on the permeability constant against the acoustic pressure and the slope from best-fit was found to be 0.039 ± 0.005 min^− 1/MPa and 0.018 ± 0.005 min^− 1/MPa, respectively. In addition, the pressure threshold for successfully induced BBBD was confirmed to be 0.4–0.6 MPa. Finally, we identified two types of leakage kinetics (fast and slow) that exhibit distinct permeability constants and temporal disruption onsets, as well as demonstrated their correlations with the applied acoustic pressure and vessel diameter. Direct assessment of vascular permeability and insights on its dependency on acoustic pressure, vessel size and leakage kinetics are important for treatment strategies of BBBD-based drug delivery.     

The Sonic hedgehog signaling pathway involved in the expression of RACK1 in the pulmonary microvascular endothelial cells induced by lipopolysaccharide北大核心CSCD

Objective To explore the effect of expression of protein kinase C receptor l(RACKl) induced by lipopolysaccharide (LPS) on Sonic hedgehog(SHH) signaling pathway in rat puhnonary microvascular endothelial cells (RPMVEC). Methods The healthy male SPF grade SD rat with 100-120 g body weight were gotten from the laboratory animal center of Annui province. Using immunocytochemistry method, the expression of RACK 1 protein in RPMVECs was detected, cultured RPMVECs were randomly divided into different groups as LPS dose-dependent group, SAG(smoothened Agonist, a SHH signaling pathway specific agonist) dose-dependent group, LPS time-dependent group, SAG time-dependent group and LPS+SAG group. In LPS dose-dependent groups, RPMVECs were cultured with 0.1, 1, 10 mg/L LPS for 8 h. In LPS time-dependent groups, RPMVECs were cultured with 10 mg/L LPS for 0, 2, 4, 8, 12, 24 h. In SAG dose-dependent groups, RPMVECs were cultured with 0.1, 1, 10 u, mol/L for 8 h. In SAG time-dependent groups, RPMVECs were cultured with 1 u, mol/L SAG for 0, 2, 4, 8, 12, 24 h. In LPS+SAG group, RPMVECs were cultured with 1 u. mol/L SAG 8 h after 10 mg/L LPS treatment for 1 h. In addition, blank group, LPS group and SAG group were set for control. Western blot were used to detect the level of RACK1 and RT-PCR were used to detect the expression of GLI-1 mRNA after intervention. Results Immunocytochemistry revealed that RACK1 were present in RPMVEC. 1. In LPS dose-dependent groups (0, 0.1, 1, 10 mg/L), the level of RACK 1 elevated as LPS dose increased correspondingly with inter-group difference (P<0.05); the relative expression levels of GLI-1 mRNA were (1.109 ± 0.063), (1.039 ± 0.135), (0.813 ± 0.066), (0.770 ± 0.105), (1 mg/L vs. 10 mg/L, P>0.05; the rest P<0.05). In LPS time-dependent groups, the relative expression level of RACK1 at 2 h (0.370 ± 0.010) was higher than that at 0 h (0.329 ± 0.008), peaked at 12 h (1.296 ± 0.048), and compared with 0 h, there was significant differences (F=l 272.204, P<0.05). The relative expression level of GLI-1 mRNA was decreased at 2 h (0.929 ± 0.007), and compared with 0 h(1.089 ± 0.042), there was significant differences (F=306.609, /><0.05). 2. In SAG dose-dependent groups, there was no significant difference in level of RACK1 between groups(all P>0.05). The relative expression levels of GLI-1 mRNA were (1.109 ± 0.063), (1.169±0.052), (3.468 ±0.128), (3.434±0.054), (0 μ.mol/L vs. 0.1 μ.mol/L and l μmol/L vs. 10 μ.mol/L, P>0.05, the rest P<0.05). Among SAG time-dependent groups, there was no significant difference in levels of RACK1 protein(P>0.05). The relative expression level of GLI-1 mRNA increased at 2 h (3.027 ± 0.065), and compared with 0 h (2.651 ± 0.123), there was significant differences (F= 132.841, P<0.05). 3. In LPS+SAG intervention groups, the expression of RACK1 was lower than that in LPS group (0.831 ± 0.040 vs. 1.189 ± 0.149, P<0.05), and the expression of GLI-1 mRNA was higher than that in LPS group (2.720 ± 0.130 vs. 0.796 ± 0.082, P<0.05). Conclusions The LPS up-regulates the expression of RACK 1 in RPMVECs, and the activated SHH signaling pathway can down-regulate the expression of RACK 1 induced by LPS in RPMVECs. © 2018 Chinese Medical Association. All rights reserved.     

The G protein–coupled receptor P2RY8 and follicular dendritic cells promote germinal center confinement of B cells,whereas S1PR3 can contribute to their dissemination

The orphan Gα13-coupled receptor P2RY8 is mutated in human germinal center (GC)–derived lymphomas and was recently found to promote B cell association with GCs in a mouse model. Here we establish that P2RY8 promotes clustering of activated B cells within follicles in a follicular dendritic cell (FDC)–dependent manner. Although mice lack a P2RY8 orthologue, we show that mouse GC B cell clustering is also dependent on FDCs acting to support the function of a Gα13-coupled receptor. Mutations in GNA13 and its downstream effector ARHGEF1 are associated with the development of disseminated GC-derived lymphomas. We find that egress of Gna13 mutant GC B cells from lymph nodes in the mouse depends on sphingosine-1-phosphate receptor-3. These findings provide evidence that FDCs promote GC confinement of both human and mouse GC B cells via Gα13-dependent pathways, and they show that dissemination of Gα13-deficient GC B cells additionally requires an egress-promoting receptor.Germinal centers (GCs) are organized as discrete, tightly confined clusters of GC B cells and T follicular helper cells (Tfh cells) in the center of reactive (secondary) follicles. Confinement of GC B cells to the GC is important for fostering interactions with antigen-loaded follicular DCs (FDCs) and Tfh cells during the selection events necessary for antibody affinity maturation (Victora and Nussenzweig, 2012). The sphingosine-1-phosphate receptor S1pr2 is a G protein–coupled receptor (GPCR) that is highly up-regulated on GC B cells and signals via the G protein Gα13 and the RhoGEF Arhgef1 (also known as p115 RhoGEF or Lsc) to inhibit cell migration in response to chemoattractants (Green et al., 2011; Muppidi et al., 2014). S1P is abundant in lymph and blood, rapidly degraded in interstitial fluids by the action of membrane phosphatases and an intracellular lyase, and thought to exist in a decaying gradient from the outer to the center follicle (Green et al., 2011; Cyster and Schwab, 2012). S1pr2 promotes GC B cell and Tfh cell confinement to the follicle center, most likely by inhibiting migration into regions of high S1P (Green et al., 2011; Moriyama et al., 2014; Muppidi et al., 2014). Maintenance of the follicular S1P gradient appears to occur in the absence of FDCs (Wang et al., 2011). However, S1pr2 deficiency does not lead to a complete dispersal of GC B cells or to their appearance in circulation, suggesting that additional factors act to promote GC B cell confinement.Deficiency in Gα13 and Arhgef1 in the mouse is also associated with loss of local GC B cell confinement, and these deficiencies lead to appearance of GC B cells in lymph and blood (Muppidi et al., 2014). The more penetrant effect of Gα13 deficiency than S1pr2 deficiency led us to identify a second human receptor, P2RY8, that can act via Gα13 to promote B cell association with GCs (Muppidi et al., 2014). Although human P2RY8 is active when expressed in the mouse, the P2RY8 gene is located on a portion of the pseudoautosomal region of the X chromosome that is lost in rodents, and no orthologues have been identified in the mouse. Because Gα13 signaling in B cells inhibits migration, the GC clustering activity of P2RY8 suggests that the P2RY8 ligand might, like S1P, be more abundant in the outer follicle than in the follicle center. Although the identity of the P2RY8 ligand is unknown, in this study, we take advantage of the ability of P2RY8 overexpression to direct cell movements in the mouse to dissect cellular requirements for controlling ligand distribution.Loss of function mutations of S1PR2, P2RY8, GNA13 (encoding Gα13), and ARHGEF1 are frequently found in human lymphomas derived from GC B cells (GC B cell–like diffuse large B cell lymphoma [GCB-DLBCL] and Burkitt lymphoma [BL]; Morin et al., 2011; Lohr et al., 2012; Schmitz et al., 2012; Muppidi et al., 2014). These malignancies are considered systemic diseases in humans, and spread of the malignant GC B cells to distant sites such as BM is associated with poor prognosis (Sehn et al., 2011). It is currently unclear whether Gα13 deficiency itself is sufficient to promote GC B cell egress or whether loss of Gα13 signaling allows other promigratory signals to dominate and promote egress.In this study, we sought to further define how GC B cells are normally confined to the GC niche and to understand how GC B cells carrying mutations typical of GC lymphomas undergo systemic spread. We demonstrate that FDCs are required for the function of human P2RY8 in promoting cell clustering at the center of mouse lymphoid follicles. Although P2RY8 is not present in the mouse, we find that FDCs act via a Gα13-dependent mechanism to promote clustering of mouse GC B cells and prevent their egress into lymph. Finally, we show that egress of Gα13-deficient mouse GC B cells is promoted by the promigratory S1P receptor S1pr3.     

The route of priming influences the ability of respiratory virus–specific memory CD8+ T cells to be activated by residual antigen

After respiratory virus infections, memory CD8⁺ T cells are maintained in the lung airways by a process of continual recruitment. Previous studies have suggested that this process is controlled, at least in the initial weeks after virus clearance, by residual antigen in the lung-draining mediastinal lymph nodes (MLNs). We used mouse models of influenza and parainfluenza virus infection to show that intranasally (i.n.) primed memory CD8⁺ T cells possess a unique ability to be reactivated by residual antigen in the MLN compared with intraperitoneally (i.p.) primed CD8⁺ T cells, resulting in the preferential recruitment of i.n.-primed memory CD8⁺ T cells to the lung airways. Furthermore, we demonstrate that the inability of i.p.-primed memory CD8⁺ T cells to access residual antigen can be corrected by a subsequent i.n. virus infection. Thus, two independent factors, initial CD8⁺ T cell priming in the MLN and prolonged presentation of residual antigen in the MLN, are required to maintain large numbers of antigen-specific memory CD8⁺ T cells in the lung airways.In recent years, there has been considerable progress in understanding the mechanisms regulating the tissue-specific migration of lymphocytes to peripheral sites. An evolving concept is that environmental factors at the site of initial priming induce the expression of tissue-selective homing molecules on activated lymphocytes. In support of this, numerous studies have demonstrated a pivotal role for antigen-presenting cells in the programming of lymphocyte trafficking patterns during priming (Mora et al., 2003; Iwata et al., 2004; Sigmundsdottir et al., 2007). In contrast, several recent studies suggest a pliable property of memory T cells in terms of their tissue tropism. Adoptive transfer and parabiosis studies have shown that the location of initial priming has little impact on the ability of circulating effector memory T cells (T_EMs) to migrate to different nonlymphoid sites (Klonowski et al., 2004; Masopust et al., 2004). One explanation for this pleotropic homing ability is that activated CD8⁺ T cells disseminate from LNs draining the site of infection to distant LNs, where they acquire additional tissue-homing molecules associated with the local microenvironment (Liu et al., 2006). Moreover, the migration of circulating central memory T cells (T_CMs) to nonlymphoid tissues also results in functional and phenotypic conversion to tissue-resident T_EM phenotype (Laouar et al., 2005, 2007; Kohlmeier et al., 2007; Marzo et al., 2007). Together, these studies demonstrate that the site of initial priming, the continued maturation of activated T cells in nondraining lymphoid tissues, and the local environment within nonlymphoid tissues all contribute the migratory properties of memory CD8⁺ T cells.Studies in both humans and mice have shown that substantial numbers of T_EM persist in the lung airways after the resolution of respiratory virus infections. The numbers of T_EM in the lung airways gradually decline over the first 6 mo after infection and then stabilize as a relatively small population of memory T cells that is maintained in the lung airways indefinitely (Ostler et al., 2001; Hogan et al., 2001a; Wiley et al., 2001; de Bree et al., 2005; van Panhuys et al., 2005). This decline and stabilization in the number of memory T cells in the lung airways correlates with a progressive decline in cell-mediated protection from a secondary challenge (Liang et al., 1994; Kündig et al., 1996; Hogan et al., 2001b; Ray et al., 2004; Bachmann et al., 2005a,b). Unlike memory T cell populations that reside in other anatomical locations, lung airway memory T cells are not directly maintained through cytokine-driven homeostatic proliferation within the lung airways. Rather, antigen-specific memory T cells present in the lung airways represent a dynamic population that is maintained by continual recruitment from the systemic memory T cell pool under steady-state conditions (Ely et al., 2006). The accumulation of memory T cells in the airways under steady-state conditions is determined by migration from the circulation and cell death within the airways, a process which we refer to as continual recruitment. A recent study has demonstrated that residual antigen is maintained in the local draining LNs for several months after respiratory virus infection, and it has suggested a model in which recent stimulation by residual antigen is required for continual recruitment of memory CD8⁺ T cells to the airways (Zammit et al., 2006). In addition, we previously demonstrated that systemic memory CD8⁺ T cells generated after a respiratory virus infection could migrate to the airways in the absence of cognate antigen, albeit at low levels (Kohlmeier et al., 2007). However, it is not known how the route of priming impacts the ability of these antigen-dependent and -independent mechanisms to promote the recruitment of memory CD8⁺ T cells to the lung airways.To better understand the mechanisms regulating the continual recruitment of memory CD8⁺ T cells to the lung airways, we investigated the localization of memory CD8⁺ T cells that had been elicited by intranasal (i.n.) versus i.p. infection. The data show that i.n.-primed memory CD8⁺ T cells were preferentially recruited and maintained in the lung airways compared with i.p.-primed CD8⁺ T cells, and the defective recruitment of i.p.-primed memory CD8⁺ T cells to the lung airways was not corrected by the presence of cognate residual antigen in the mediastinal LN (MLN). Importantly, the ability of virus-specific memory CD8⁺ T cells to be activated by residual antigen in the MLN was restricted to i.n.-primed cells, and this activation resulted in multiple phenotypic changes which are associated with lung airway-resident cells. Collectively, the data suggest that not only the prolonged presentation of cognate antigen in the MLN but also T cell priming in the LNs that drain the respiratory tract during the primary response are required for the continual recruitment of memory CD8⁺ T cells to the lung airways.     

The Recognition of the Nonclassical Major Histocompatibility Complex (MHC) Class I Molecule,T10, by the γδ T Cell,G8

Recent studies have shown that many nonclassical major histocompatibility complex (MHC) (class Ib) molecules have distinct antigen-binding capabilities, including the binding of nonpeptide moieties and the binding of peptides that are different from those bound to classical MHC molecules. Here, we show that one of the H-2T region–encoded molecules, T10, when produced in Escherichia coli, can be folded in vitro with β₂-microglobulin (β₂m) to form a stable heterodimer in the absence of peptide or nonpeptide moieties. This heterodimer can be recognized by specific antibodies and is stimulatory to the γδ T cell clone, G8. Circular dichroism analysis indicates that T10/β₂m has structural features distinct from those of classical MHC class I molecules. These results suggest a new way for MHC-like molecules to adopt a peptide-free structure and to function in the immune system.Classical MHC class I (class Ia) molecules possess a highly specialized groove occupied by short peptides that are acquired inside the cell during MHC heterodimer assembly. This peptide–MHC interaction not only contributes to the stability of the heterodimer on the cell surface, but forms the basis for its function, as complexes of intracellular pathogen derived peptides with MHC are the ligands for cytolytic αβ T cells. Recently, many nonclassical MHC class I molecules, such as those encoded in the Q, T, M, and CD1 regions, have been found to possess binding properties different from those of classical MHC molecules (as reviewed in references 1 and 2). These studies suggest that class Ib molecules have evolved for specific tasks that are distinct from those of classical class I MHC. For example, M3 and human CD1 have been proposed to play a special role in controlling microbial infection by binding and presenting N-formylated peptide and lipid antigens, respectively (3–7). Murine CD1 molecules have been shown to bind hydrophobic peptides and are thought to stimulate regulatory αβ T cells (8, 9). Some Qa molecules were found to bind mixtures of peptides with molecular properties different from those bound to classical MHC molecules (10, 11). Other Qa molecules have been suggested to have roles in the generation of regulatory T cells, as well as the elimination of bacterially infected cells (12, 13).Although the majority of the cells that respond to class Ib ligands bear the αβ TCR, the H-2T–encoded T10 and the closely related T22 (94% identity) proteins were first identified as the ligands for two γδ T cells, KN6 and G8 (14– 16). G8 was generated by immunizing BALB/c nude mice with B10.BR spleen cells (17), whereas KN6 was derived from a C57BL/6 double-negative thymocyte (18). Attempts to derive αβ T cells specific for these molecules, using either cells naturally expressing T10/T22 or transfected with these genes as immunogen, have been unsuccessful (reference 19; Schild, H., and Y.-h. Chien, unpublished data). Analysis of the recognition of T10/T22 by G8 shows it to be clearly different from MHC class I recognition by αβ T cells. In particular, G8 can respond to stimulator cells that lack functional peptide-loading mechanisms for either MHC class I or class II molecules (15, 16, 19). All variations in the ability of different stimulator cells to activate G8 can be attributed solely to the level of T10/T22 surface expression. In addition, G8 is able to respond to T10/T22 expressed on Drosophila melanogaster cells, which inherently lack peptide-loading machinery and therefore express MHC molecules that are devoid of peptide (20). Together, these experiments indicated that T10/T22 may not present peptide for its recognition by G8.In this study, we evaluate directly whether components other than the T10/T22 heavy chain and β₂-microglobulin (β₂m) are necessary for its recognition and structural stability. We find that Escherichia coli–produced T10 and β₂m can be folded in vitro in the absence of peptide or nonpeptide moieties. This is in contrast with classical class I MHC molecules, whose folding of E. coli–produced heavy chain and β₂m can take place only in the presence of an appropriate peptide (21, 22). The reconstituted T10/β₂m heterodimer is biochemically homogeneous and can be recognized by specific antibodies and the G8 γδ T cell. The far-UV circular dichroic (CD)¹ spectrum of T10/β₂m is different from that of typical MHC class I molecules. These data suggest that T10 may have evolved to possess distinctive structural features capable of carrying out a specialized function in the immune system.     

Specific induction of the 70-kD heat stress proteins by the tyrosine kinase inhibitor herbimycin-A protects rat neonatal cardiomyocytes. A new pharmacological route to stress protein expression?

Heat shock protein (hsp) induction by stressful stimuli such as heat and ischemia is known to protect cardiac cells from severe stress. The ability to induce hsp's in the heart directly by "nonstressful" means would potentially have important clinical implications. In noncardiac cells, the tyrosine kinase inhibitor herbimycin-A has been shown to induce the 72-kD hsp. We therefore examined whether herbimycin-A and another tyrosine kinase inhibitor, genistein, could induce 70-kD hsp's in primary cultures of rat neonatal cardiomyocytes, and whether these treatments protect against severe stress. Primary cardiomyocytes were incubated with herbimycin-A or genistein. hsp induction was measured 16-20 h later by Western blotting. Cell survival after subsequent lethal heat stress or simulated ischemia was assessed using trypan blue exclusion and released lactate dehydrogenase activity. Our results indicate that, in cardiac cells, herbimycin-A induces 70-kD hsp's but not hsp90, -60, -25, or glucose-regulated protein 78, whereas genistein has no effect on hsp's. Moreover, hsp induction correlated with the ability of herbimycin-A to protect cells against severe stress, whereas genistein has no protective effects. This suggests that herbimycin-A may induce 70-kD hsp's via a tyrosine kinase-independent mechanism. These results indicate the possibility of a pharmacological approach to HSP70 induction and cardiac protection, which may ultimately be of clinical relevance.     

The evaluation of autoantibodies against oxidatively modified low-density lipoprotein (LDL), susceptibility of LDL to oxidation,serum lipids and lipid hydroperoxide levels,total antioxidant status,antioxidant enzyme activities,and endothelial dysfunction in patients with Behçet's disease

OBJECTIVES: Beh?et's disease is a multisystem disorder characterized by a chronic inflammation including acute attacks and remission periods. Decreased enzyme activity of the antioxidant system and increased levels of free radicals may have important roles in the damage of tissues observed in the disease period. In addition, the atherogenic tendency of serum lipid, lipoproteins, lipid peroxidation levels and endothelial dysfunction accompany the above mentioned findings. As a consequence of these events, different degrees of low density lipoprotein (LDL) oxidation occur in vivo, and then autoantibodies against oxidized-LDL(AuAb-oxLDL) are produced. DESIGN AND METHODS: Lipids, lipoproteins, lipid hydroperoxide, AuAb-oxLDL, total antioxidant status (TAS), serum-soluble intercellular adhesion molecule-1 (sICAM-1), plasminogen activator inhibitor 1 (PAI-1) levels in serum, the activities of antioxidant enzymes including glutathione peroxidase (GSH-Px), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in erythrocytes and plasma, were determined in 25 patients with Beh?et's disease and in 25 healthy volunteers. Also, susceptibility to copper-induced in vitro oxidation of LDL by using lag time, a measure of resistance to oxidation, oxidation rate and extent of oxidation, a measure of diene production in both groups, was studied. RESULTS: It was observed that lipid hydroperoxide and AuAb-oxLDL levels in patients with Beh?et's disease were significantly higher, but erythrocyte SOD, CAT, plasma GSH-Px activities, and TAS were significantly lower than those in healthy subjects. Susceptibility of LDL to oxidation in the patients was found to be increased. Total cholesterol, LDL-C and apo B levels and acute phase reactants were significantly higher, but HDL-C and apo AI levels were significantly lower, in patients when compared to healthy subjects. The levels of AuAb-oxLDL in patients were found to correlate with TAS, total cholesterol, LDL-C, lipid hydroperoxide and erythrocyte SOD activities (r = -0.62, p < 0.01; r = 0.64, p < 0.01; r = 0.55, p < 0.01; r = 0.81, p < 0.01; r = -0.63, p < 0.01, respectively). In addition, lipid hydroperoxide levels were found to correlate with total cholesterol, LDL-C and erythrocyte SOD activities (r = 0.45, p < 0.05; r = 0.45, p < 0.05; r = -0.46, p < 0.05, respectively). PAI-1 and sICAM-1 were found to be increased in the patients and correlated with AuAb-oxLDL and lipid hydroperoxide levels (r = 0.56, p < 0.01; r = 0.67, p < 0.01 and r = 0.59, p < 0.01; r = 0.61, p < 0.01, respectively). CONCLUSIONS: It was concluded that the observed increase of lipid, lipoproteins, lipid hydroperoxide, susceptibility of LDL to oxidation, autoantibodies against ox-LDL levels and decrease of antioxidant enzyme activities and total antioxidant status and increased secretion of endothelial derivated peptides including sICAM and PAI-1, and their interactions may indicate that there is a tendency to atherothrombotic events in patients with Beh?et's disease.