The effect of selective phosphodiesterase inhibitors,alone and in combination,on a murine model of allergic asthma

BackgroundThe anti-inflammatory effects of the selective phosphodiesterase (PDE) inhibitors cilostazol (PDE 3), RO 20-1724 (PDE 4) and sildenafil (PDE 5) were examined in a murine model of allergic asthma. These compounds were used alone and in combination to determine any potential synergism, with dexamethasone included as a positive control.MethodsControl and ovalbumin sensitised Balb/C mice were administered orally with each of the possible combinations of drugs at a dose of 3 mg/Kg for 10 days.ResultsWhen used alone, RO 20-1724 significantly reduced eosinophil influx into lungs and lowered tumour necrosis factor-α, interleukin-4 and interleukin-5 levels in the bronchoalveolar lavage fluid when compared to untreated mice. Treatment with cilostazol or sildenafil did not significantly inhibit any markers of inflammation measured. Combining any of these PDE inhibitors produced no additive or synergistic effects. Indeed, the anti-inflammatory effects of RO 20-1724 were attenuated by co-administration of either cilostazol or sildenafil.ConclusionsThese results suggest that concurrent treatment with a PDE 3 and/or PDE 5 inhibitor will reduce the anti-inflammatory effectiveness of a PDE 4 inhibitor.     

T-cell activation without proliferation in juvenile idiopathic arthritis

A study was done to determine if the differentiation and activation phenotype of T cells in synovial fluid (SF) from patients with juvenile idiopathic arthritis (JIA) is associated with T-cell proliferation in situ. Mononuclear cells were isolated from 44 paired samples of peripheral blood and SF. Differentiation and activation markers were determined on CD4 and CD8 T cells by flow cytometry. Cell-cycle analysis was performed by propidium iodide staining, and surface-marker expression was also assessed after culture of the T cells under conditions similar to those found in the synovial compartment. The majority of the T cells in the SF were CD45RO⁺CD45RB^dull. There was greater expression of the activation markers CD69, HLA-DR, CD25 and CD71 on T cells from SF than on those from peripheral blood. Actively dividing cells accounted for less than 1% of the total T-cell population in SF. The presence or absence of IL-16 in T-cell cultures with SF or in a hypoxic environment did not affect the expression of markers of T-cell activation. T cells from the SF of patients with JIA were highly differentiated and expressed early and late markers of activation with little evidence of in situ proliferation. This observation refines and extends previous reports of the SF T-cell phenotype in JIA and may have important implications for our understanding of chronic inflammation.     

Class I patatin基因5‘侧翼区驱动的GUS基因在马铃薯块茎中专一性表达

Binary vectors pPATIs (with partial signal sequence) and pPATI (without signal sequence) were constructed by fusing 1.4 kb 5' flanking regions of Class I patatin gene with GUS. Transient GUS expression was observed in in vitro tuber slices bombarded with pPATI. These constructs were then introduced into potato (cv. Desiree) via Agrobacterium tumefaciens transformation. Transgenic potato plants were confirmed by X-Gluc staining and PCR. Using in vitro tuberization system, GUS activities were assayed by fluorescence. It was shown that, in plants transformed with PATI-GUS, GUS specific activities were about 10-20 fold higher in tubers than in stems. Increased sucrose concentration could not induce PATI-GUS expression, but light enhanced PATI-GUS expression in cultured shoots.     

Functional importance of polar and charged amino acid residues in transmembrane helix 14 of multidrug resistance protein 1 (MRP1/ABCC1): identification of an aspartate residue critical for conversion from a high to low affinity substrate binding state

Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs.     

Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter

The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.     

Role of alpha-helical conformation of histatin-5 in candidacidal activity examined by proline variants

Human salivary histatin-5 (Hsn-5) is a potent in vitro anticandidal agent. The aim of this study was to investigate the importance of alpha-helical structure of Hsn-5 for its candidacidal activity. The following three Hsn-5 variants, where one or more functionally nonessential residues were replaced with proline (potent alpha-helix breaker), were produced by Escherichia coli expression system: H21P (1P), H19P/H21P (2P), and E16P/H19P/H21P (3P). The activities of purified proteins were determined by candidacidal assays, and the secondary structures by circular dichroism (CD) spectroscopy in trifluoroethanol (TFE) that is considered the helix-promoting solvent, and lysophosphatidyl-glycerol (LPG) micelles, the environment that more closely resembles the biological membranes. Our results indicated that 3P variant displayed a candidacidal activity which was similar to that of unaltered Hsn-5 (0P), while 1P and 2P variants showed lower cidal activity. The CD spectra in TFE indicated that 3P variant has less helical characteristics than the 0P, 1P and 2P. These results suggested that the alpha-helical content of Hsn-5 proline variants does not correlate with the candidacidal activity. Further, the CD spectral analysis of peptides in LPG micelles indicated the formation of beta-turn structures in 0P and 3P variants. In conclusion, 3P variant which exhibited comparable candidacidal activity to 0P contains lower percentage of alpha-helical structure than 1P and 2P variants, which exhibited lower candidacidal activity. This suggests alpha-helix may not be important for anticandidal activity of Hsn-5.     

Deciphering membrane protein structures from protein sequences

ABSTRACT: Co-evolving positions within protein sequences have been used as spatial constraints to develop a computational approach for modeling membrane protein structures.     

A potentially common peptide target in secreted heat shock protein-90α for hypoxia-inducible factor-1α-positive tumors

Deregulated accumulation of hypoxia-inducible factor-1α (HIF-1α) is a hallmark of many solid tumors. Directly targeting HIF-1α for therapeutics is challenging. Our finding that HIF-1α regulates secretion of heat shock protein-90α (Hsp90α) for cell migration raises the exciting possibility that targeting the secreted Hsp90α from HIF-1α-positive tumors has a better clinical outlook. Using the HIF-1α-positive and metastatic breast cancer cells MDA-MB-231, we show that down-regulation of the deregulated HIF-1α blocks Hsp90α secretion and invasion of the cells. Reintroducing an active, but not an inactive, HIF-1α into endogenous HIF-1α-depleted cells rescues both Hsp90α secretion and invasion. Inhibition of Hsp90α secretion, neutralization of secreted Hsp90α action, or removal of the cell surface LRP-1 receptor for secreted Hsp90α reduces the tumor cell invasion in vitro and lung colonization and tumor formation in nude mice. Furthermore, we localized the tumor-promoting effect to a 115-amino acid region in secreted Hsp90α called F-5. Supplementation with F-5 is sufficient to bypass the blockade of HIF-1α depletion and resumes invasion by the tumor cells under serum-free conditions. Because normal cells do not secrete Hsp90α in the absence of stress, drugs that target F-5 should be more effective and less toxic in treatment of HIF-1α-positive tumors in humans.