PPP3CB Inhibits Cell Proliferation and the Warburg Effect in Bladder Cancer by Blocking PDHK1.

BACKGROUND: Cancer treatment has recently shifted towards metabolic approaches aimed at enhancing therapeutic efficacy. Somewhat surprisingly, a known regulator of energy metabolism in normal tissues, PPP3CB, is down-regulated in bladder cancer. This suggests that PPP3CB could exert an inhibitory effect on bladder cancer through its role in energy metabolism. METHODS: To explore the above hypothesis, we employed non-targeted metabolism screening in bladder cancer cells with knockdown of PPP3CB. Glucose uptake and lactate production were carefully measured using specialized assay kits for glucose/lactic acid content. Western blot analysis was also used to evaluate the expression levels of pyruvate dehydrogenase kinase 1 (PDHK1) and p-PDHA1 in cells with PPP3CB knockdown. To substantiate the findings, co-immunoprecipitation (co-IP) experiments were performed to validate the interaction between PPP3CB and PDHK1. Various in vitro assays were also performed, including clone formation assay and Cell Counting Kit-8 (CCK8) viability assays. The in vivo anti-tumor potential of PPP3CB in bladder cancer was also studied using a nude mouse tumorigenesis model. RESULTS: Significant down-regulation of PPP3CB was observed in bladder tumors, and potent anti-tumor effects of PPP3CB were observed in vitro. Investigation of the underlying mechanism by which PPP3CB hampers glycolysis in bladder cancer cells revealed that it interacted with PDHK1 to inhibit its protein stabilization. PDHK1 thus appears to be a crucial mediator through which PPP3CB exerts its inhibitory effects on bladder cancer cells. CONCLUSIONS: In summary, PPP3CB exerts strong inhibitory influences on bladder cancer cell proliferation and glycolysis via its destabilization of PDHK1. These results highlight the potential of PPP3CB as a novel regulator of the Warburg effect. Interestingly, the downregulation of PPP3CB in bladder cancer cells increases the Warburg effect, thereby generating more lactic acid and reshaping the tumor microenvironment so as to promote tumor cell proliferation.

A comparison of invasive arterial blood pressure measurement with oscillometric non-invasive blood pressure measurement in patients with sepsis.

PURPOSE: This study aimed to compare non-invasive oscillometric blood pressure (NIBP) measurement with invasive arterial blood pressure (IBP) measurement in patients with sepsis. METHODS: We conducted a retrospective study to evaluate the agreement between IBP and NIBP using the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. Paired blood pressure measurements of mean arterial pressure (MAP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were compared using Bland-Altman analysis and paired Student's t test. We also focus on the effect of norepinephrine (NE) on the agreement between the two methods and the association between blood pressure and mortality during intensive care unit (ICU) stay. RESULTS: A total of 96,673 paired blood pressure measurements from 6060 unique patients were analyzed in the study. In Bland-Altman analysis, the bias (± SD, 95% limits of agreement) was 6.21 mmHg (± 12.05 mmHg, - 17.41 to 29.83 mmHg) for MAP, 0.39 mmHg (± 19.25 mmHg, - 37.34 to 38.12 mmHg) for SBP, and 0.80 mmHg (± 12.92 mmHg, - 24.52 to 26.12 mmHg) for DBP between the two techniques. Similarly, large limits of agreement were shown in different groups of NE doses. NE doses significantly affected the agreement between IBP and NIBP. SBP between the two methods gave an inconsistent assessment of patients' risk of ICU mortality. CONCLUSION: IBP and NIBP were not interchangeable in septic patients. Clinicians should be aware that non-invasive MAP was clinically and significantly underestimated invasive MAP.

Surgical therapy and survival in young patients with stage I-II hepatocellular carcinoma: a retrospective cohort study.

Background: Hepatocellular carcinoma (HCC) is regarded as a high-mortality cancer, but the effectiveness of surgical strategies for young patients with early-stage HCC remains controversial. We aimed to analyze the survival in young patients with stage I-II HCC who underwent different kinds of surgical treatments. Methods: Overall survival (OS) and cancer-specific survival (CSS) were compared among patients aged 18-45 years with stage I-II HCC from the Surveillance, Epidemiology, and End Results (SEER) database (2004-2013) who underwent local tumor destruction (LTD), wedge or segmental resection (WSR), lobectomy resection (LR), liver transplantation (LT), or non-surgery. Univariate and multivariate analyses and Kaplan-Meier method were used to examine the OS and CSS of the patients. A stratification analysis of CSS was also conducted among the subgroups. Results: Data from 664 patients were extracted. The median survival time was 46 months. In the multivariate analysis of OS, compared with non-surgery, LTD [hazard ratio (HR), 0.37; 95% confidence interval (CI): 0.25-0.54; P<0.0001], LR (HR, 0.29; 95% CI: 0.19-0.45; P<0.0001), and WSR (HR, 0.26; 95% CI: 0.17-0.39; P<0.0001) had better outcomes, and LT had the best survival benefit (HR, 0.24; 95% CI: 0.16-0.36; P<0.0001), which was similar to CSS. In the stratification analysis, compared with the non-surgery group, among patients with chemotherapy, LT reduced the risk of CSS by 64% (HR, 0.36; 95% CI: 0.19-0.66; P interaction=0.0004). Conclusions: Surgery offers a survival benefit compared with non-surgery for young patients with stage I-II HCC. LT is associated with better survival than WSR, LR, and LTD.

Albuca Bracteate Polysaccharides Synergistically Enhance the Anti-Tumor Efficacy of 5-Fluorouracil Against Colorectal Cancer by Modulating ß-Catenin Signaling and Intestinal Flora.

The first-line treatment for colorectal cancer (CRC) is 5-fluorouracil (5-FU). However, the efficacy of this treatment is sometimes limited owing to chemoresistance as well as treatment-associated intestinal mucositis and other adverse events. Growing evidence suggests that certain phytochemicals have therapeutic and cancer-preventing properties. Further, the synergistic interactions between many such plant-derived products and chemotherapeutic drugs have been linked to improved therapeutic efficacy. Polysaccharides extracted from Albuca bracteata (Thunb.) J.C.Manning and Goldblatt (ABP) have been reported to exhibit anti-oxidant, anti-inflammatory, and anti-tumor properties. In this study, murine CRC cells (CT26) and a murine model of CRC were used to examine the anti-tumor properties of ABP and explore the mechanism underlying the synergistic interactions between ABP and 5-FU. Our results revealed that ABP could inhibit tumor cell proliferation, invasion, and migratory activity in vitro and inhibited tumor progression in vivo by suppressing ß-catenin signaling. Additionally, treatment with a combination of ABP and 5-FU resulted in better outcomes than treatment with either agent alone. Moreover, this combination therapy resulted in the specific enrichment of Ruminococcus, Anaerostipes, and Oscillospira in the intestinal microbiota and increased fecal short-chain fatty acid (SCFA) levels (acetic acid, propionic acid, and butyric acid). The improvement in the intestinal microbiota and the increase in beneficial SCFAs contributed to enhanced therapeutic outcomes and reduced the adverse effects of 5-FU. Together, these data suggest that ABP exhibits anti-neoplastic activity and can effectively enhance the efficacy of 5-FU in CRC treatment. Therefore, further research on the application of ABP in the development of novel anti-tumor drugs and adjuvant compounds is warranted and could improve the outcomes of CRC patients.

Profiles of expression pattern and tissue distribution of host defense peptides genes in different chicken (Gallus gallus) breeds related to body weight.

Host defense peptides (HDPs) are an important first line of defense with antimicrobial and immunomodulatory properties. Selection for increased body weight is hypothesized to be related to reduced immune response. We studied the relationships among body weight, age, and the HDP expression patterns in intestine and immune organs. We used chickens with marked differences of body sizes. The non-selected Daweishan mini chickens showed the highest indexes of immune organs and the lowest concentrations of the plasma immune parameters C3, C4, IgA, and IgY, while the commercial Avian broiler showed the opposite results. The Daweishan mini chickens showed the highest mRNA expressions of HDP genes in small intestine followed by the semi-selected Wuding chickens. Compared with local breeds, broiler chickens showed higher mRNA expression of HDP genes in spleen, thymus, and bursa. Body weight and HDP expression levels were negatively correlated in the intestine and positively in the immune organs. Our results indicated that the HDP immune regulatory roles in small intestine acted as first line of defense in innate immunity in local breeds, and as an adaptive immunity in broiler chickens. Selection was associated with different expression expressions of HDP genes in breed-, age-, and organ-specific manners.

De Novo Designed Amphipathic α-Helical Antimicrobial Peptides Incorporating Dab and Dap Residues on the Polar Face To Treat the Gram-Negative Pathogen, Acinetobacter baumannii.

We have designed de novo and synthesized ten 26-residue D-conformation amphipathic α-helical cationic antimicrobial peptides (AMPs), seven with "specificity determinants", which provide specificity for prokaryotic cells over eukaryotic cells. The ten AMPs contain five or six positively charged residues (d-Arg, d-Lys, d-Orn, l-Dab, or l-Dap) on the polar face to understand their role in hemolytic activity against human red blood cells and antimicrobial activity against seven Acinetobacter baumannii strains, resistant to polymyxin B and colistin, and 20 A. baumannii worldwide isolates from 2016 and 2017 with antibiotic resistance to 18 different antibiotics. AMPs with specificity determinants and with l-Dab and l-Dap residues on the polar face have essentially no hemolytic activity at 1000 µg/mL (380 µM), showing for the first time the importance of these unusual amino acid residues in solving long-standing hemolysis issues of AMPs. Specificity determinants maintained excellent antimicrobial activity in the presence of human sera.

Design of Novel Amphipathic α-Helical Antimicrobial Peptides with No Toxicity as Therapeutics against the Antibiotic-Resistant Gram-Negative Bacterial Pathogen, Acinetobacter Baumannii.

We designed de novo and synthesized two series of five 26-residue amphipathic α-helical cationic antimicrobial peptides (AMPs) with five or six positively charged residues (D-Lys, L-Dab (2,4-diaminobutyric acid) or L-Dap (2,3-diaminopropionic acid)) on the polar face where all other residues are in the D-conformation. Hemolytic activity against human red blood cells was determined using the most stringent conditions for the hemolysis assay, 18h at 37°C, 1% human erythrocytes and peptide concentrations up to 1000 µg/mL (~380 µM). Antimicrobial activity was determined against 7 Acinetobacter baumannii strains, resistant to polymyxin B and colistin (antibiotics of last resort) to show the effect of positively charged residues in two different locations on the polar face (positions 3, 7, 11, 18, 22 and 26 versus positions 3, 7, 14, 15, 22 and 26). All 10 peptides had two D-Lys residues in the center of the non-polar face as "specificity determinants" at positions 13 and 16 which provide specificity for prokaryotic cells over eukaryotic cells. Specificity determinants also maintain excellent antimicrobial activity in the presence of human sera. This study shows that the location and type of positively charged residue (Dab and Dap) on the polar face are critical to obtain the best therapeutic indices.

Separation of highly charged (+5 to +10) amphipathic α-helical peptide standards by cation-exchange and reversed-phase high-performance liquid chromatography.

We are currently examining the potential of amphipathic cationic α-helical peptides as a new generation of peptide standards for both cation-exchange high-performance liquid chromatography and reversed-phase chromatography. Thus, amphipathic peptides are particularly suitable for high-performance liquid chromatography standards due to the preferred binding of the non-polar face to the hydrophobic stationary phase of reversed-phase packings or the preferred binding of the polar face to the charged/hydrophilic stationary phase of cation-exchange packings. The ability of different reversed-phase or cation-exchange matrices to separate mixtures of peptide standards with only subtle hydrophilicity/hydrophobicity variations in both the non-polar and polar face of the peptides can then be assessed. Currently, we have designed de novo a mixture of six 26-residue all D-conformation amphipathic cationic α-helical peptides with a single, positively charged lysine residue in the center of the non-polar face and an increasing number of lysine residues (4-9 residues) replacing neutral residues in the polar face, resulting in an overall net positive charge of +5 to +10. Thus, the non-polar, preferred reversed-phase chromatography binding face remains constant, with only the polar face varying in hydrophilicity/hydrophobicity. Interestingly, even with the non-polar face remaining constant, reversed-phase columns of varying functional group properties (e.g., C8, C18, phenyl, polar endcapped, polar embedded) and porosity (porous versus superficially porous) were able to separate the six peptides in aq. TFA/acetonitrile gradients, albeit with different selectivities. The value of the standards in cation-exchange chromatography was expressed by monitoring the requirement of acetonitrile (0-40% in the mobile phase) to overcome hydrophobic interactions of the peptides with the cation-exchange matrix matrix when eluting with sodium perchlorate gradients at pH 6.5. Interestingly, the resolution of the higher charged peptides (+8,+9,+10) was particularly sensitive to acetonitrile levels. Our results clearly demonstrate the excellent potential of these novel peptide standards to enable optimal column choice and mobile phase conditions for reversed-phase chromatography and cation-exchange chromatography for peptide separations.

Role of positively charged residues on the polar and non-polar faces of amphipathic α-helical antimicrobial peptides on specificity and selectivity for Gram-negative pathogens.

We have designed de novo and synthesized eight 26-residue all D-conformation amphipathic α-helical cationic antimicrobial peptides (AMPs), four with "specificity determinants" which provide specificity for prokaryotic cells over eukaryotic cells and four AMPs without specificity determinants. The eight AMPs contain six positively charged Lys residues on the polar face in four different arrangements to understand the role of these residues have on antimicrobial activity against 14 Acinetobacter baumannii strains, seven of which were resistant to polymyxin B and colistin; six diverse Pseudomonas aeruginosa strains and 17 Staphylococcus aureus strains, nine of which were methicillin-sensitive, and eight of which were methicillin-resistant. The four AMPs without specificity determinants are extremely hemolytic. In contrast, the four AMPs with specificity determinants had dramatic improvements in therapeutic indices showing the importance of specificity determinants in removing eukaryotic cell toxicity. The specificity determinants combined with the location of positively charged residues on the polar face provide Gram-negative pathogen selectivity between A. baumannii and S. aureus. Specificity determinants maintain excellent antimicrobial activity in the presence of human sera, whereas the AMPs without specificity determinants were inactive. This study clearly shows the potential of amphipathic α-helical AMPs with specificity determinants as therapeutics to replace existing antibiotics.

Satellite-based entanglement distribution over 1200 kilometers.

Long-distance entanglement distribution is essential for both foundational tests of quantum physics and scalable quantum networks. Owing to channel loss, however, the previously achieved distance was limited to ~100 kilometers. Here we demonstrate satellite-based distribution of entangled photon pairs to two locations separated by 1203 kilometers on Earth, through two satellite-to-ground downlinks with a summed length varying from 1600 to 2400 kilometers. We observed a survival of two-photon entanglement and a violation of Bell inequality by 2.37 ± 0.09 under strict Einstein locality conditions. The obtained effective link efficiency is orders of magnitude higher than that of the direct bidirectional transmission of the two photons through telecommunication fibers.

Platform technology to generate broadly cross-reactive antibodies to α-helical epitopes in hemagglutinin proteins from influenza A viruses.

We have utilized a de novo designed two-stranded α-helical coiled-coil template to display conserved α-helical epitopes from the stem region of hemagglutinin (HA) glycoproteins of influenza A. The immunogens have all the surface-exposed residues of the native α-helix in the native HA protein of interest displayed on the surface of the two-stranded α-helical coiled-coil template. This template when used as an immunogen elicits polyclonal antibodies which bind to the α-helix in the native protein. We investigated the highly conserved sequence region 421-476 of HA by inserting 21 or 28 residue sequences from this region into our template. The cross-reactivity of the resulting rabbit polyclonal antibodies prepared to these immunogens was determined using a series of HA proteins from H1N1, H2N2, H3N2, H5N1, H7N7, and H7N9 virus strains which are representative of Group 1 and Group 2 virus subtypes of influenza A. Antibodies from region 449-476 were Group 1 specific. Antibodies to region 421-448 showed the greatest degree of cross-reactivity to Group 1 and Group 2 and suggested that this region has a great potential as a "universal" synthetic peptide vaccine for influenza A. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 144-159, 2016.

"Specificity Determinants" Improve Therapeutic Indices of Two Antimicrobial Peptides Piscidin 1 and Dermaseptin S4 Against the Gram-negative Pathogens Acinetobacter baumannii and Pseudomonas aeruginosa.

A new class of antimicrobial agents with lower rates of resistance and different targets is urgently needed because of the rapidly increasing resistance to classical antibiotics. Amphipathic cationic α-helical antimicrobial peptides (AMPs) represent such a class of compounds. In our previous studies, using a 26-residue de novo designed antimicrobial peptide, we proposed the concept of "specificity determinant(s)": positively charged residue(s) in the center of the non-polar face of AMPs that could decrease hemolytic activity/toxicity but increase or maintain the same level of antimicrobial activity to increase dramatically the therapeutic index. In the current study, we used d-enantiomers of two AMPs, Piscidin 1 isolated from fish and dermaseptin S4 isolated from frog. We substituted different positions in the center of the hydrophobic face with one or two lysine residue(s) (one or two "specificity determinant(s)"). This simple modification not only maintained or improved antimicrobial activity against Gram-negative pathogens Acinetobacter baumannii (11 strains) and Pseudomonas aeruginosa (6 strains), but also dramatically decreased hemolytic activity of human red blood cells, as predicted. Therapeutic indices improved by 55-fold and 730-fold for piscidin 1 (I9K) and dermaseptin S4 (L7K, A14K), respectively, against A. baumannii. Similarly, the therapeutic indices improved 32-fold and 980-fold for piscidin 1 (I9K) and dermaseptin S4 (L7K, A14K), respectively, against P. aeruginosa.

An improved approach to hydrophilic interaction chromatography of peptides: salt gradients in the presence of high isocratic acetonitrile concentrations.

Hydrophilic interaction chromatography (HILIC) for separations of peptides has been employed infrequently, particularly considering that this technique was introduced over 20 years ago. The present manuscript describes a radical departure from the traditional HILIC elution approach, where separations are achieved via increasing salt (sodium perchlorate) gradients in the presence of high isocratic concentrations (>80%) of acetonitrile, denoted HILIC/SALT. This initial study compared to reversed-phase chromatography (RPC), HILIC and HILIC/SALT for the separation of mixtures of synthetic peptide standards varying in structure (amphipathic α-helix, random coil), length (10-26 residues), number of positively charged residues (+1 to +11) and hydrophilicity/hydrophobicity. Results showed a marked superiority of the HILIC/SALT approach compared to traditional HILIC and excellent complementarity to RPC for peptide separations. We believe these initial results offer a new dimension to HILIC, enabling it to transform from an occasional HPLC approach for peptide separations to a more generally applicable method.

Rational design of α-helical antimicrobial peptides to target Gram-negative pathogens, Acinetobacter baumannii and Pseudomonas aeruginosa: utilization of charge, 'specificity determinants,' total hydrophobicity, hydrophobe type and location as design parameters to improve the therapeutic ratio.

The rapidly growing problem of increased resistance to classical antibiotics makes the development of new classes of antimicrobial agents with lower rates of resistance urgent. Amphipathic cationic α-helical antimicrobial peptides have been proposed as a potential new class of antimicrobial agents. The goal of this study was to take a broad-spectrum, 26-residue, antimicrobial peptide in the all-D conformation, peptide D1 (K13) with excellent biologic properties and address the question of whether a rational design approach could be used to enhance the biologic properties if the focus was on Gram-negative pathogens only. To test this hypothesis, we used 11 and 6 diverse strains of Acinetobacter baumannii and Pseudomonas aeruginosa, respectively. We optimized the number and location of positively charged residues on the polar face, the number, location, and type of hydrophobe on the non-polar face and varied the number of 'specificity determinants' in the center of the non-polar face from 1 to 2 to develop four new antimicrobial peptides. We demonstrated not only improvements in antimicrobial activity, but also dramatic reductions in hemolytic activity and unprecedented improvements in therapeutic indices. Compared to our original starting peptide D1 (V13), peptide D16 had a 746-fold improvement in hemolytic activity (i.e. decrease), maintained antimicrobial activity, and improved the therapeutic indices by 1305-fold and 895-fold against A. baumannii and P. aeruginosa, respectively. The resulting therapeutic indices for D16 were 3355 and 895 for A. baumannii and P. aeruginosa, respectively. D16 is an ideal candidate for commercialization as a clinical therapeutic to treat Gram-negative bacterial infections.

Anti-tuberculosis activity of α-helical antimicrobial peptides: de novo designed L- and D-enantiomers versus L- and D-LL-37.

With the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (Mtb), a new class of antimycobacterial agents with very different modes of action compared to classical antibiotics, are urgently needed. In this study, a series of 26-residue, amphipathic, α-helical antimicrobial peptides consisting of all D-amino acid residues and synthetic human L-LL37 (L-enantiomer) and D-LL37 (D-enantiomer) were investigated against M. tuberculosis susceptible strain (H37Rv) and a clinical multi-drug resistant strain (Vertulo). Minimal inhibitory concentrations (MICs) were determined through a peptide killing assay. D5, the most active analog against M. tuberculosis had a MIC value of 11.2 µM (35.2 µg/ml) against H37Rv strain and 15.6 µM (49 µg/ml) against the MDR strain. Peptide D1 had similar activity as D5 against the MDR strain (57 µg/mL), a 9-fold improvement in hemolytic activity and a 7.4-fold better therapeutic index compared to D5. Surprisingly, LL37 enantiomers showed little to no activity compared to the de-novo designed α-helical antimicrobial peptides.

Effects of hydrophobicity on the antifungal activity of alpha-helical antimicrobial peptides.

We utilized a series of analogs of D-V13K (a 26-residue amphipathic alpha-helical antimicrobial peptide, denoted D1) to compare and contrast the role of hydrophobicity on antifungal and antibacterial activity to the results obtained previously with Pseudomonas aeruginosa strains. Antifungal activity for zygomycota fungi decreased with increasing hydrophobicity (D-V13K/A12L/A20L/A23L, denoted D4, the most hydrophobic analog was sixfold less active than D1, the least hydrophobic analog). In contrast, antifungal activity for ascomycota fungi increased with increasing hydrophobicity (D4, the most hydrophobic analog was fivefold more active than D1). Hemolytic activity is dramatically affected by increasing hydrophobicity with peptide D4 being 286-fold more hemolytic than peptide D1. The therapeutic index for peptide D1 is 1569-fold and 62-fold better for zygomycota fungi and ascomycota fungi, respectively, compared with peptide D4. To reduce the hemolytic activity of peptide D4 and improve/maintain the antifungal activity of D4, we substituted another lysine residue in the center of the non-polar face (V16K) to generate D5 (D-V13K/V16K/A12L/A20L/A23L). This analog D5 decreased hemolytic activity by 13-fold, enhanced antifungal activity to zygomycota fungi by 16-fold and improved the therapeutic index by 201-fold compared with D4 and represents a unique approach to control specificity while maintaining high hydrophobicity in the two hydrophobic segments on the non-polar face of D5.

Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides.

In our previous study, we utilized a 26-residue amphipathic alpha-helical antimicrobial peptide L-V13K (Chen et al., Antimicrob Agents Chemother 2007, 51, 1398-1406) as the framework to study the effects of peptide hydrophobicity on the mechanism of its antimicrobial action. In this study, we explored the effects of net charge and the number of positively charged residues on the hydrophilic/polar face of L-V13K on its biological activity (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity, helicity, and peptide self-association). The net charge of V13K analogs at pH 7 varied between -5 and +10 and the number of positively charged residues varied from 1 to 10. The minimal inhibitory concentrations (MIC) against six strains of Pseudomonas aeruginosa as well as other gram-negative and gram-positive bacteria were determined along with the maximal peptide concentration that produces no hemolysis of human red blood cells (MHC). Our results show that the number of positively charged residues on the polar face and net charge are both important for both antimicrobial activity and hemolytic activity. The most dramatic observation is the sharp transition of hemolytic activity on increasing one positive charge on the polar face of V13K i.e., the change from +8 to +9 resulted in greater than 32-fold increase in hemolytic activity (250 microg/ml to <7.8 microg/ml, respectively).

Expression of intracellular domain of epidermal growth factor receptor and generation of its monoclonal antibody.

To prepare monoclonal antibody specific to epidermal growth factor receptor (EGFR) intracellular domain, its gene was amplified from total RNA of A431 cell by RT-PCR. Then the gene was cloned into prokaryotic vector pET30a(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni2+-NTA agarose. Then the anti-EGFR monoclonal antibody (mAb) was prepared with classical hybridoma technique. Positive clones were selected using indirect enzyme-linked immunoabsorbent assay (ELISA). Totally 4 hybridoma clones were obtained and these mAbs were IgG1 (3 clones) and IgG2a (1 clone), respectively. Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line. The mAbs will be useful in the study of EGFR-mediated signal transduction.

Isolation of acetylcholinesterase from apoptotic human lung fibroblast cells by antibody affinity chromatography.

Acetylcholinesterase (AChE; EC3.1.1.7) is well known for its role in the hydrolysis of acetylcholine at cholinergic synapses to terminate neurotransmission. In addition to its synaptic presence, AChE has been found to be in non-cholinergic cells such as hematopoietic and osteogenic cells. We have recently reported that AChE is expressed in various cells undergoing apoptosis. To characterize AChE in apoptotic cells and to investigate the role of AChE expression in apoptosis, we devised a method to purify AChE expressed in apoptotic human lung fibroblast cell line HLF. The isolation of this enzyme is mainly based on inhibitor ligand affinity chromatography using immobilized tacrine. However, this method is only effective in isolating active AChE. Here we employed antibody-based chromatography and found that both active and inactive AChE were present in apoptotic HLF cells. Active AChE was predominantly observed in the nuclei of apoptotic cells, while inactive AChE was mainly present in the cytoplasm. Therefore, our method provides an opportunity to investigate further the role of AChE, especially inactive AChE, in apoptosis.