The pharmacological mechanism of Xiaoyaosan polysaccharide reveals improvement of CUMS-induced depression-like behavior by carbon source-triggered butyrate-producing bacteria.

AIMS: Here, regulatory effects of Xiaoyaosan polysaccharide on entire intestinal flora and butyrate-producing bacteria were investigated to reveal their pharmacological mechanism serving as bacterial-derived carbon sources for regulating intestinal microecology during the treatment of chronic unpredictable mild stress (CUMS)-induced depression in rats. METHODS AND RESULTS: The effects were measured by analyzing depression-like behavior, intestinal flora, butyrate-producing bacteria diversity, and fecal butyrate content. After intervention, CUMS rats exhibited alleviated depression and increased body weight, sugar water consumption rate, and performance index in the open-field test (OFT). The abundance of dominant phyla, such as Firmicutes and Bacteroidetes, and dominant genera, such as Lactobacillus and Muribaculaceae, was regulated to restore the diversity and abundance of the entire intestinal flora to a healthy level. The polysaccharide enriched the diversity of butyrate-producing bacteria, increased the abundance of the butyrate-producing bacteria Roseburia sp. and Eubacterium sp., reduced the abundance of Clostridium sp., increased the distribution of Anaerostipes sp., Mediterraneibacter sp., and Flavonifractor sp., and subsequently increased the content of butyrate in the intestine. CONCLUSIONS: These findings suggest that the Xiaoyaosan polysaccharide alleviates unpredictable mild stress-induced depression-like chronic behavior in rats by regulating the composition and abundance of the entire intestinal flora, restoring the diversity of butyrate-producing bacteria, and increasing the butyrate levels.

Mediastinal Lymphadenopathy in the National Lung Screening Trial (NLST) Is Associated with Interval Lung Cancer.

Background There are currently no evidence-based guidelines for the management of enlarged mediastinal lymph nodes found on lung cancer screening (LCS) CT scans. Purpose To assess the frequency and clinical significance of enlarged mediastinal lymph nodes on the initial LCS CT scans in National Lung Screening Trial (NLST) participants. Materials and Methods A retrospective review of the NLST database identified all CT trial participants with at least one enlarged (≥1.0 cm) mediastinal lymph node identified by site readers on initial CT scans. Each study was reviewed independently by two thoracic radiologists to measure the two largest nodes and to record morphologic characteristics. Scans with extensively calcified mediastinal lymph nodes or nodes measuring less than 1 cm were excluded. Frequency and time to lung cancer diagnosis, lung cancer stage, and histologic findings were compared between NLST participants with and without lymphadenopathy. Results Of the 26 722 NLST participants, 422 (1.6%) had enlarged noncalcified mediastinal lymph nodes on the initial LCS CT scan. Mediastinal lymphadenopathy was associated with an increase in lung cancer cases (72 of 422 participants [17.1%; 95% CI: 13.6, 21.0] vs 1017 of 26 300 [3.9%; 95% CI: 3.6, 4.1]; P < .001), earlier diagnosis (restricted mean survival time ± standard error, 2285 days ± 44 vs 2611 days ± 2; P < .001), the presence of lung nodules (P < .001), advanced stage at presentation (22 of 72 participants [31%] with cancer at stage IIIA vs 410 of 1017 [40.3%] at stage IA; P < .001), and increased mortality (P < .001). The majority of participants with lung cancers in the LCS group with mediastinal lymphadenopathy were detected at initial LCS CT (50 of 422 participants [11.8%; 95% CI: 8.9, 15.3] vs T1-T7, 22 of 422 [5.3%; 95% CI: 3.3, 7.8]; P < .001). There was no association between mediastinal lymphadenopathy and lung cancer histologic findings, CT appearance, or location of lung nodules (P > .05 based on unadjusted pairwise association analyses). Conclusion Noncalcified mediastinal lymphadenopathy in the low-dose lung cancer screening study sample was associated with an increase in lung cancer, an earlier diagnosis, more advanced-stage disease, and increased mortality. More aggressive treatment of these patients appears warranted. © RSNA, 2021 Online supplemental material is available for this article. See also the editorials by McLoud and by Mascalchi and Zompatori in this issue.

Radiofrequency ablation-increased CXCL10 is associated with earlier recurrence of hepatocellular carcinoma by promoting stemness.

Radiofrequency ablation (RFA) represents a valuable choice in hepatocellular carcinoma (HCC); however, local recurrence of HCC is common after RFA. Here, 20 primary HCC patients treated by RFA were enrolled. Before (termed 0d) and after RFA treatment for 1 and 7 days (termed 1d and 7d, respectively), plasma and noncancerous tissue were collected. ELISA assay showed that plasma C-X-C motif chemokine 10 (CXCL10) was increased in ten patients (type I patients) but decreased in the other 10 patients (type II patients). The mean interval for HCC recurrence in type I patients was less than the mean interval in type II patients. Interestingly, a significant negative correlation between interval for HCC recurrence and fold change of plasma CXCL10 (1d/0d or 7d/0d) was identified, suggesting that RFA-induced CXCL10 is associated with earlier HCC recurrence. Immunofluorescence assay showed that the receptor of CXCL10, chemokine (C-X-C motif) receptor 3 (CXCR3), was significantly increased in type I, but not type II, patients after RFA. In vitro assay demonstrated that CXCL10 stimulus increased the rate of CD133(+) cancer stem cells (CSCs) in HepG2 cells by binding to CXCR3 and then inducing c-Myc expression. Many studies have reported that induction of CD133(+) CSCs contributes to HCC recurrence. Thus, CXCL10-increased CD133(+) CSCs by activating CXCR3/c-Myc pathway might accelerate HCC recurrence after RFA. These data might have potential implications for HCC therapy.

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells.

Pancreatic beta cells have well-developed ER to accommodate for the massive production and secretion of insulin. ER homeostasis is vital for normal beta cell function. Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by 1D SDS-PAGE coupled with HPLC-MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments. GO analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. Further bioinformatics analysis also identified potential beta cell specific ER proteins as well as ER proteins present in the risk genetic loci of type 2 diabetes. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes-causing conditions. All MS data have been deposited in the ProteomeXchange with identifier PXD001081 (http://proteomecentral.proteomexchange.org/dataset/PXD001081).

Asymmetric transmission and optical rotation of a quasi-3D asymmetric metallic structure.

Three-dimensional (3D) asymmetric plasmonic structures possessing asymmetric optical transmission properties have been widely studied. However, these structures have limitations for application due to fabrication techniques. Here, a quasi-3D asymmetric structure built up by a metallic rod-shaped particles layer and a metallic L-shaped holes layer was fabricated by the sputtering and the focused ion beam (FIB) milling. A broadband (1000-1600 nm) asymmetric transmission and optical rotation have been demonstrated experimentally. Numerical calculations show that the coupling between the cavity and particle plasmonic resonances contributes to this effect.

A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-ß2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-ß2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.

Kruppel like factor 16 promotes lung adenocarcinoma progression by upregulating lamin B2.

Lung cancer is one of the most common causes of cancer-related death. In the past decade, the treatment and diagnosis of lung cancer have progressed significantly in early efforts to promote the survival of lung cancer patients. Kruppel like factor 16 (KLF16) is a zinc finger transcription factor that regulates a diverse array of developmental events and cellular processes. KLF16 is involved in the progression of various cancer types. However, the role of KLF16 in the development of lung cancer remains unknown. In this study, KLF16 was overexpressed in lung cancer samples. KLF16 downregulation inhibited lung cancer cell proliferation and migration. Conversely, KLF16 overexpression promoted lung cancer cell growth and invasion. Mechanistically, the expression level LMNB2 was suppressed by KLF16 knockdown and was promoted by KLF16 overexpression. The overall survival of patients with high LMNB2 levels was poor. Luciferase assays showed that KLF16 promoted the transcription activity of LMNB2 gene. Concomitantly, the expression level of LMNB2 was also higher in lung adenocarcinoma (LUAD) than in normal tissues, and its knockdown or overexpression can reverse the effect of KLF16 overexpression or knockdown on lung cancer cell proliferation, migration, and even tumorigenesis, indicating that LMNB2 also functions as an oncogene. In conclusion, KLF16 can be used as a potential therapeutic and preventive biomarker in lung cancer treatment and prognosis by actively regulating the expression of LMNB2.

Clinical Significance of Lung-RADS Category 3 Lesions in the National Lung Screening Trial.

INTRODUCTION: To determine the clinical significance of category 3 (CAT3) abnormalities and the necessity of a 6-month follow-up computed tomography (CT). We also explored features associated with increased lung cancer risk. METHODS: From the National Lung Screening Trial database, we identified participants with CAT3 lesions at prevalence screen. Rates of lung cancer and lung cancer-specific deaths (LSDs) were compared between those who underwent first follow-up CT before 6 months (early diagnostic group) and those who underwent annual screening (annual diagnostic group). We estimated the change in LSD if the 6-month CT was eliminated. Regression analysis was performed to identify features associated with participants with CAT3 who developed lung cancer. RESULTS: A total of 1763 CAT3s were identified (6.6% of all participants who had low-dose CT), with 108 lung cancers (6.1%) and 41 LSDs (2.3%) in a 7-year period. Rates of lung cancer (7.5% versus 3.1%) and LSD (4.0% versus 1.0%) were higher in the early diagnostic group than in the annual diagnostic group. We estimated an increase in LSD of 0.6% of all participants with CAT3 (24.4% of all LSDs) if the 6-month CT was not performed. Multivariate regression analysis found that increased age, emphysema, and a part-solid nodule greater than 5 mm were associated with participants with CAT3 who developed lung cancer. CONCLUSIONS: CAT3 lesions are uncommon, and eliminating the 6-month CT would potentially increase LSD by 0.6% of all patients with CAT3. Age, emphysema, and part-solid nodule greater than 5 mm may be useful in risk prediction models to determine which participants with CAT3 are more likely to develop lung cancer and suggest which patients may need more intense follow-up.

Community quarantine strategy against coronavirus disease 2019 in Anhui: An evaluation based on trauma center patients.

OBJECTIVE: The objective of our study was to introduce community quarantine strategy against coronavirus disease 2019 (COVID-19) in Anhui and evaluate the effectiveness of community quarantine based on trauma center (TC) patients. METHOD: The structure of community quarantine strategy was illustrated. Distribution of injuries among patients in two TCs between January 24, 2020 and February 24, 2020 was described. Multiple linear regression was used to analyze the correlation between the distribution of Injuries in TCs and the number of COVID-19-associated cases. RESULTS: A total of 757 TC patients in the two hospitals were enrolled. The number of traffic injuries and outdoor injuries showed a significant decrease in the early stage and began to increase on February 17. The number of indoor injuries neither decreased nor increased. Multiple linear regression analysis revealed a significant correlation between COVID-19-associated cases and traffic and outdoor injuries. CONCLUSION: From the perspective of the injuries in TCs, community quarantine strategy was effectively implemented and significantly slowed the outbreak of COVID-19 in Anhui. However, the implementation and maintenance of the strategy is costly and requires the participation of the entire population.

Specificity proteins Sp1 and Sp3 interact with the rat GTP cyclohydrolase I proximal promoter to regulate transcription.

The role of the proximal promoter GC-box in regulating basal and cAMP-dependent GTP Cyclohydrolase I gene transcription was investigated using a variety of cell lines and techniques. These studies show that the GC-box is composed of a triad of cis-elements that in vitro bind specificity proteins Sp1 and Sp3. Sp1 and Sp3 were found associated with the native proximal promoter in PC12 cells but were not recruited to the promoter during cAMP-dependent transcription. Studies using Drosophila SL2 cells showed that Sp3 occupies two sites within the GC-box and enhances transcription when acting alone and synergistically when combined with nuclear factor-Y (NF-Y) and CCAAT/Enhancer-Binding Protein (C/EBP)beta, cognate binding proteins for the adjacent cAMP response element (CRE) and CCAAT-box cAMP response elements. In contrast, Sp1 bound only one site within the GC-box and did not enhance transcription unless combined with NF-Y and C/EBPbeta. Studies in SL2 cells also showed that Sp1 and Sp3 do not co-occupy the GC-box, and accordingly Sp1 competes for Sp3 binding to repress Sp3-dependent transcription. In PC12 cells, complete mutation of the GC-box reduced basal but not cAMP-dependent transcription, resulting in an overall increase in the cAMP response and demonstrating that formation of this enhanceosome does not require Sp1 or Sp3. Experiments in which the GC-box was replaced with a Gal4 element and the promoter challenged with Gal4 fusion proteins support this conclusion and a role for Sp3 in maintaining high levels of basal transcription in PC12 cells. Equivalent amounts of Sp1 and Sp3 were found associated with the native proximal promoter in PC12 and Rat2 cells, which differ 10-fold in basal transcription. Similar levels of methylation of CpG dinucleotides located within the GC-box were also observed in these two cells lines. These results suggest that Sp1 and Sp3 bound to the GC-box might help to preserve an open chromatin configuration at the proximal promoter in cells which constitutively express low levels of GTP Cyclohydrolase I.

[Progress and challenges in optical cochlear implant].

Optical cochlear implant has been occuring as a new cochlear implant which utilizes laser pulses to stimulate hearing. Compared to electronic cochlear implant, it has demonstrated higher spatial selectivity and less radiation scattering, which could lead to higher fidelity cochlear prostheses. At present, most investigations have focused on experiments in vivo. Although a lot of exciting results have been obtained, the mechanisms of laser stimulation is still open. In this paper, a brief review on the recent new findings of optical cochlear implant is given, and possible mechanisms are discussed. In the end, new experimental proposals are suggested which could help to explore the mechanisms of laser-cochlea stimulation.

[Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris].

Human placental ribonuclease inhibitor is an acidic protein of Mr approximately 50 kDa with unusually high contents of leucine and cysteine. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. HRI has 32 cysteine residues, and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates HRI. The most proximal cysteine residues in native HRI are two pairs that are adjacent in sequence. In the present paper, two molecules of alanine to substitute for cys328/cys329 were performed by site-directed mutagenesis. The site-mutated RI cDNA was constructed into plasmid pPIC9K, and then transformed Pichia pastoris GS115 by electroporation. After colony screening , the bacterium was cultured and the product was purified with affinity chromatography. The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect. The results indicated that there was no much change in the affinity for RNase A detected when compared with the wild type of RI. But the capacity of anti-oxidative effect was increased by 7-9 times. The enhance in anti-oxidative effect might be the reason for preventing the formation of disulfide bond between cys328 and cys329 and the three dimensional structure of RI was thereby maintained.

A critical role of CXCR2 PDZ-mediated interactions in endothelial progenitor cell homing and angiogenesis.

Bone marrow-derived endothelial progenitor cells (EPCs) contribute to neovessel formation in response to growth factors, cytokines, and chemokines. Chemokine receptor CXCR2 and its cognate ligands are reported to mediate EPC recruitment and angiogenesis. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif which has been reported to modulate cellular signaling and functions. Here we examined the potential role of the PDZ motif in CXCR2-mediated EPC motility and angiogenesis. We observed that exogenous CXCR2 C-tail significantly inhibited in vitro EPC migratory responses and angiogenic activities, as well as in vivo EPC angiogenesis. However, the CXCR2 C-tail that lacks the PDZ motif (ΔTTL) did not cause any significant changes of these functions in EPCs. In addition, using biochemical assays, we demonstrated that the PDZ scaffold protein NHERF1 specifically interacted with CXCR2 and its downstream effector, PLC-ß3, in EPCs. This suggests that NHERF1 might cluster CXCR2 and its relevant signaling molecules into a macromolecular signaling complex modulating EPC cellular functions. Taken together, our data revealed a critical role of a PDZ-based CXCR2 macromolecular complex in EPC homing and angiogenesis, suggesting that targeting this complex might be a novel and effective strategy to treat angiogenesis-dependent diseases.

The relationship between serum interleukins and T-lymphocyte subsets in patients with severe acute respiratory syndrome.

OBJECTIVES: To observe the changes of serum interleukins (IL), T-lymphocyte subsets, and white blood cell (WBC) count in patients with severe acute respiratory syndrome (SARS), and to investigate the relationship between injured immune function, immune response and disturbed immune adjustment in SARS patients. METHODS: The levels of serum IL-2, IL-10, IL-12 and T-lymphocyte subset counts were measured in 35 clinically diagnosed SARS patients by using enzyme linked immunosorbant assay (ELISA). The relationship between the measured results and WBC count was further analyzed. RESULTS: The level of serum IL was increased to a great extent in the 35 SARS patients, and the levels of serum IL-2, IL-10 and IL-12 were 242.53 (92.69) pg/ml, 77.43 (63.37) pg/ml and 65.94 (43.21) pg/ml, respectively. The level of serum IL-2 increased markedly (P < 0.01). The peripheral blood CD(3)(+), CD(4)(+) and CD(8)(+) counts were lower than normal in 23 patients (67.7%), 26 patients (74.3%) and 15 patients (42.9%), respectively. The peripheral blood WBC counts were lower than 4.0 x 10(9)/L in 10 patients, and their CD(3)(+), CD(4)(+) and CD(8)(+) counts were 583.90 (315.58) x 10(6)/L, 272.00 (94.13) x 10(6)/L and 209.00 (72.21) x 10(6)/L, respectively. The peripheral blood WBC counts were (4.0 - 10.0) x 10(9)/L in 20 patients, and their CD(3)(+), CD(4)(+) and CD(8)(+) counts were 700.00 (502.96) x 10(6)/L, 347.00 (247.58) x 10(6)/L and 322.05 (228.47) x 10(6)/L, respectively. The peripheral blood WBC counts were higher than 10.0 x 10(9)/L in 5 patients, and their CD(3)(+), CD(4)(+) and CD(8)(+) counts were 1466.00 (630.86) x 10(6)/L, 783.00 (311.14) x 10(6)/L and 640.00 (294.40) x 10(6)/L, respectively. The decreased CD(3)(+), CD(4)(+) and CD(8)(+) counts were consistent with the decreased WBC counts. The level of IL in SARS patients was significantly higher than that in patients with chronic hepatitis B (P < 0.01). CONCLUSIONS: The level of serum IL is closely related to cell immunity in SARS patients. The level of serum IL is increased evidently while CD(3)(+), CD(4)(+) and CD(8)(+) counts decrease. Both serum IL and CD are associated with injury of immune function, and thus they could be regarded as a monitoring index for judging the condition of SARS patients and prescribing immune therapy.

Structural insights into neutrophilic migration revealed by the crystal structure of the chemokine receptor CXCR2 in complex with the first PDZ domain of NHERF1.

Neutrophil plays an essential role in host defense against infection, but uncontrolled neutrophilic infiltration can cause inflammation and severe epithelial damage. We recently showed that CXCR2 formed a signaling complex with NHERF1 and PLC-2, and that the formation of this complex was required for intracellular calcium mobilization and neutrophilic transepithelial migration. To uncover the structural basis of the complex formation, we report here the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal sequence of CXCR2 at 1.16 Å resolution. The structure reveals that the CXCR2 peptide binds to PDZ1 in an extended conformation with the last four residues making specific side chain interactions. Remarkably, comparison of the structure to previously studied PDZ1 domains has allowed the identification of PDZ1 ligand-specific interactions and the mechanisms that govern PDZ1 target selection diversities. In addition, we show that CXCR2 can bind both NHERF1 PDZ1 and PDZ2 in pulldown experiments, consistent with the observation that the peptide binding pockets of these two PDZ domains are highly structurally conserved. The results of this study therefore provide structural basis for the CXCR2-mediated neutrophilic migration and could have important clinical applications in the prevention and treatment of numerous neutrophil-dependent inflammatory disorders.

CXCR2 macromolecular complex in pancreatic cancer: a potential therapeutic target in tumor growth.

The signaling mediated by the chemokine receptor CXC chemokine receptor 2 (CXCR2) plays an important role in promoting the progression of many cancers, including pancreatic cancer, one of the most lethal human malignancies. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif at its carboxyl termini, which might interact with potential PDZ scaffold/adaptor proteins. We have previously reported that CXCR2 PDZ motif-mediated protein interaction is an important regulator for neutrophil functions. Here, using a series of biochemical assays, we demonstrate that CXCR2 is physically coupled to its downstream effector phospholipase C-ß3 (PLC-ß3) that is mediated by PDZ scaffold protein Na(+)/H(+) exchange regulatory factor 1 (NHERF1) into a macromolecular signaling complex both in vitro and in pancreatic cancer cells. We also observe that disrupting the CXCR2 complex, by gene delivery or peptide delivery of exogenous CXCR2 C-tail, significantly inhibits the biologic functions of pancreatic cancer cells (i.e., proliferation and invasion) in a PDZ motif-dependent manner. In addition, using a human pancreatic tumor xenograft model, we show that gene delivery of CXCR2 C-tail sequence (containing the PDZ motif) by adeno-associated virus type 2 viral vector potently suppresses human pancreatic tumor growth in immunodeficient mice. In summary, our results suggest the existence of a physical and functional coupling of CXCR2 and PLC-ß3 mediated through NHERF1, forming a macromolecular complex that is critical for efficient and specific CXCR2 signaling in pancreatic cancer progression. Disrupting this CXCR2 complex could represent a novel and effective treatment strategy against pancreatic cancer.

In vitro analysis of PDZ-dependent CFTR macromolecular signaling complexes.

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis(1-3). CFTR has been implicated in two major diseases: cystic fibrosis (CF)(4) and secretory diarrhea(5). In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States(6). Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut(7). Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo(8-19). In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP(20-27). The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)(20). Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities(22). This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells(16-18). Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay(16-19,28,29). Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR(16-19,28,29).

[Immunogenecity of cytomegalovirus-infected fibroblasts].

AIM: To analyze the capability of cytomegalovirus (CMV)-infected human embryonic lung fibroblasts (HELFs) to induce immune response. METHODS: HELFs were infected with cytomegalovirus and stained with antibody against HLA-A2 molecular, the expression of HLA-A2 was detected by FCM. The infected HELFs were incubated with individual pp65 peptide NLVPMVATV. While the uninfected and unloaded infected HELFs served as control respectively. After PBMC was added to the differently treated HELFs and incubated, the immune response was measured with IFN-gamma release as readout. RESULTS: The expression of HLA-A molecular on infected fibroblasts diminished markedly compared with that on the uninfected. The peptides expressed on the infected HELFs together with those pulsed externally induced a stronger response than the infected HELFs alone. CONCLUSION: Although CMV can down-regulate the expression of MHC I on the infected cells, it can not decrease the capacity of cells to present peptides loaded externally, and therefore still induce immune response to some extent.

Protein kinase A-dependent recruitment of RNA polymerase II, C/EBP beta and NF-Y to the rat GTP cyclohydrolase I proximal promoter occurs without alterations in histone acetylation.

Cyclic-AMP stimulation of GTP cyclohydrolase I (GCH1) gene transcription was investigated in PC12 cells, the protein kinase A-deficient PC12 cell line 126-1B2 and C6 cells using transient transfection assays of proximal promoter reporter constructs and wild type or dominant negative proteins, chromatin immunoprecipitation and real-time quantitative PCR. These studies show that protein kinase A is necessary and sufficient for cAMP-dependent transcription conferred by both the cAMP regulatory element and the adjacent CCAAT-box. In intact cells these cis-elements were shown to bind cAMP response element binding protein, CCAAT-enhancer binding protein beta and nuclear factor-Y, with each protein controlling a different aspect of the cAMP response. Cyclic-AMP acting through protein kinase A stimulated promoter recruitment of CCAAT-enhancer binding protein beta, nuclear factor-Y and RNA polymerase II while depleting the promoter of cyclic-AMP response element binding protein. Stimulation of transcription by cAMP was not associated with increased acetylation of histones H3 and H4 at proximal promoter nucleosomes, indicating that histone acetyltransferases are not involved in this response. Nonetheless, pharmacological inhibition of histone deacetylase activity did increase histone H4 acetylation and the recruitment of RNA polymerase II, indicating that histone acetyltransferases are normally associated with the proximal promoter. Only in C6 cells, however, did inhibition of histone deacetylases stimulate transcription and synergize with cAMP. These experiments provide the first glimpse of the GCH1 gene promoter functioning within intact cells and supply evidence for the involvement of histone acetyltransferase-containing complexes in GCH1 gene transcription.