In vitro activation of CD8 interphotoreceptor retinoid-binding protein-specific T cells requires not only antigenic stimulation but also exogenous growth factors

In a previous study, we demonstrated that immunization with the uveitogenic peptide interphotoreceptor retinoid-binding protein (IRBP) 1-20 induces both CD4 and CD8 uveitogenic T cells in the B6 mouse. In the current study, we determined the role of the CD8 IRBP-specific T cells in the pathogenesis of experimental autoimmune uveitis. We also determined the conditions that facilitated the activation of CD8 autoreactive T cells. Our results showed that the beta2-microglobulin(-/-) mouse had a greatly decreased susceptibility to induction of experimental autoimmune uveitis by adoptive transfer of IRBP-specific T cells from B6 mice. We also showed that unlike CD4 autoreactive T cells, activated CD8 autoreactive T cells produced only a limited number and amounts of growth factors. As a result, in the absence of exogenously supplied growth factor(s), CD8 T cell activation and expansion were aborted. However, the growth and expansion of triggered CD8 autoreactive T cells could be supported by various cytokines. In addition to factors produced by activated CD4 autoreactive T cells, factors produced by nonlymphoid cells, such as IL-7 and IL-15, and unidentified factors in the culture supernatants of astrocytes and retinal pigment epithelial cells support the CD8 autoreactive T cells as well. Finally, we showed that, although several cytokines augmented the CD8 T cell response in vitro, different cytokines appeared to act on different CD8 subsets or on different activation/differentiation phases of CD8 autoreactive T cells. As a result, cytokines, such as IL-7, supported the proliferation and survival of CD8 IRBP-specific T cells, while others had only a growth-promoting effect.     

Effects of selective estrogen receptor modulator raloxifene plus 17-beta estradiol in aorta and mammary gland of female experimental atherosclerosis rabbits and possible involvement of ERK signal transduction pathway

The present study investigated the effects of raloxifene, a second generation selective estrogen receptor modulator (SERM), plus 17-betaE2 on aortic atherosclerosis and mammary gland hyperplasia in ovariectomized, cholesterol-fed rabbits. Following 10 weeks of raloxifene, 17-betaE2, or raloxifene plus 17-betaE2 administration, serum total cholesterol, triglyceride, low density lipoprotein were significantly decreased in the drug groups compared to the placebo group. Consistent with serum lipid results, the total lesion area for each aorta of the drug groups decreased significantly as compared to the placebo group. HE staining of aorta paraffin section showed that in the drug groups the endothelial monolayer was almost continuous. While in mammary gland, HE staining of paraffin sections indicated the hyperplasia of epithelial cells (in 17-betaE2 group) was obviously inhibited in raloxifene plus 17-betaE2 group. In cultured vascular smooth muscle cell (VSMC), the results of MTT and [3H]TdR incorporation showed that the drug groups could inhibit AngII-induced proliferation of VSMC. Western blotting proved that raloxifene plus 17-betaE2 inhibited the expression of phosphorylated ERK protein, similar to 17-betaE2 but different from raloxifene. This effect was inhibited by PD98059 (inhibitor of MAPK) or ICI182780 (ER antagonist). In conclusion, this study suggests that SERM raloxifene plus 17-betaE2 improves the lipid metabolism and relieves the aorta changes of female experimental atherosclerosis rabbits, which are partly implemented by the inhibition of VSMC growth through ERK cascade. The hyperplasia of mammary gland epithelial cells could be significantly inhibited by raloxifene plus 17-betaE2.     

Abnormal modulation of cholinergic neurotransmission by endomorphin 1 and endomorphin 2 in isolated bronchus of type 1 diabetic rats

To assess whether diabetes alters the regulatory effects of mu-opioid receptor (MOR) agonists on the cholinergic bronchoconstriction, we investigated the inhibitory effects of endomorphins (EMs) on the electrical field stimulation (EFS)-induced cholinergic bronchoconstriction in type 1 diabetic rats. At 4 weeks after the onset of diabetes, both the EFS- and exogenous acetylcholine (ACh)-induced bronchoconstriction in diabetes in vitro were greater than those in non-diabetes rats. Furthermore, endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited the response to EFS in diabetic rat isolated bronchus in a concentration- and frequency-dependent manner, which is in agreement with that in non-diabetes. However, the inhibitory effects of EMs on the EFS-induced bronchoconstriction in diabetes were significantly weaker than those in non-diabetes. Both EM1 and EM2 (1 microM) had no effect on the contractile response to exogenous ACh, indicating a prejunctional effect. Furthermore, the inhibitory effect on the EFS-induced bronchoconstriction was blocked by naloxone (10 microM). Eight weeks after the induction of diabetes, both the EFS- and exogenous ACh-induced bronchoconstrictions in diabetes were further enhanced compared to those in short-time (4 weeks) diabetic rats. Moreover, the inhibitory effects of EMs on the EFS-induced bronchoconstriction were further attenuated. These results suggest that dysfunction of presynaptic inhibitory modulation through opioid receptor by EMs may take place in the bronchus of diabetic rats.     

Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 A resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A.fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A.niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A.fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.     

Novel spironolactone-analogs as impurities in spironolactone

Six novel spironolactone-analogs steroids (3-8) were isolated from spironolactone by using various chromatographic methods. Their structures were elucidated by spectrometric analysis. Two of the analogs (3 and 7) were confirmed by X-ray crystallography. The A-ring of compounds 3-7 is opened at C-2C-3 bond, and compound 7 is an organic polysulfide, which has a rare, nine-membered ring with a five sulfur atom bridge.     

Propyl paraben inhibits voltage-dependent sodium channels and protects cardiomyocytes from ischemia-reperfusion injury

The effects of propyl paraben, an antimicrobial preservative, on voltage-dependent sodium current and myocardial ischemia-reperfusion injury were investigated in isolated adult rat cardiomyocytes. Whole cell voltage-clamp recording showed that propyl paraben reversibly blocked the voltage-gated sodium channel both in concentration- and voltage-dependent manners. Propyl paraben (500 microM but not 100 microM) significantly shifted the steady-state inactivation of the sodium channel toward the hyperpolarizing direction at the V(1/2) point. Consistent with the above result, the propidium iodide (PI) uptake test revealed that pretreatment with 500 microM but not 100 microM of propyl paraben significantly reduced cell death induced by 45 min of sustained ischemia followed by 15 h of reperfusion (42.37 +/- 7.01% of cell viability in control and 71.05 +/- 7.06% in the propyl paraben group), suggesting that propyl paraben can protect myocytes from ischemia-reperfusion injury. These results indicate a possible correlation between the inhibition of sodium current and cardioprotection against ischemia-reperfusion injury.     

The protein profile of acetazolamide-treated sera in mice bearing Lewis neoplasm

The aim of the present research is to analyze the proteome of neoplasm serum before and after treated with acetazolamide (20, 40, 80 mg kg(-1) d(-1) for 3 days p.o.). The Lewis lung carcinoma mice were used and carried out a comprehensive proteomic analysis by using the technologies of high-resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS). The results showed that the acetazolamide could dramatically reduce the lung metastasis and primary tumor growth. Its most potent inhibition rate on lung metastases was reach to 77.7% at the dose of 80 mg kg(-1) d(-1). The two dimension electrophoresis and software analysis reveal 393 protein spots in control gel, 385 protein spots were detected in treated gel and matched 209 protein spots with control gel, indicating that intensive changes had occurred during the process of treatment. Two obviously different spots were cut off from gel and for the peptide mass fingerprinting. Data base searching showed the two proteins' peptide much more mach with Histone H2B fragment and Ubc-like protein CROC1 fragment. The results suggest that acetazolamide has a strong anti-tumor and anti-metastasis effect on Lewis-lung-carcinoma. The mechanism may be related to its regulation on plenty of proteins, in particular, on upregulation of H2B and CROC-1 expression of postreplicational DNA repair related protein in serum.     

A novel in vivo role for osteoprotegerin ligand in activation of monocyte effector function and inflammatory response

Osteoprotegerin Ligand (OPGL) is a member of the tumor necrosis factor ligand superfamily and has been shown to be involved in interactions between T cells and dendritic cells. Its role in monocyte effector function, however, has not been defined. In the present study a role for OPGL in activating monocytes/macrophages has been characterized. OPGL was found to up-regulate receptor activator of NF-kappaB (RANK) receptor expression on monocytes, regulate their effector function by inducing cytokine and chemokine secretion, activate antigen presentation through up-regulation of co-stimulatory molecule expression, and promote survival. This activation is mediated through the MAPK pathway as evidenced by activation of p38 and p42/44 MAPK and up-regulation of BCL-XL protein levels. A physiological role for OPGL in monocyte activation and effector function was tested in a model of lipopolysaccharide-induced endotoxic shock. Administration of receptor activator of NF-kappaB (RANK)-Fc to block OPGL activity in vivo was able to protect mice from death induced by sepsis, indicating a hitherto undescribed role for OPGL in monocyte function and in mediating inflammatory response. This was further tested in an animal model of inflammation-mediated arthritis. Treatment with RANK-Fc significantly ameliorated disease development and attenuated bone destruction. Thus, our study strongly suggests that administration of receptor fusion proteins to specifically block OPGL activity in vivo may result in blocking development of monocyte/macrophage-mediated diseases.     

Crystal structure of CD26/dipeptidyl-peptidase IV in complex with adenosine deaminase reveals a highly amphiphilic interface

Dipeptidyl-peptidase IV (DPPIV or CD26) is a homodimeric type II membrane glycoprotein in which the two monomers are subdivided into a beta-propeller domain and an alpha/beta-hydrolase domain. As dipeptidase, DPPIV modulates the activity of various biologically important peptides and, in addition, DPPIV acts as a receptor for adenosine deaminase (ADA), thereby mediating co-stimulatory signals in T-lymphocytes. The 3.0-A resolution crystal structure of the complex formed between human DPPIV and bovine ADA presented here shows that each beta-propeller domain of the DPPIV dimer binds one ADA. At the binding interface, two hydrophobic loops protruding from the beta-propeller domain of DPPIV interact with two hydrophilic and heavily charged alpha-helices of ADA, giving rise to the highest percentage of charged residues involved in a protein-protein contact reported thus far. Additionally, four glycosides linked to Asn229 of DPPIV bind to ADA. In the crystal structure of porcine DPPIV, the observed tetramer formation was suggested to mediate epithelial and lymphocyte cell-cell adhesion. ADA binding to DPPIV could regulate this adhesion, as it would abolish tetramerization.