The Distinctive Permutated Domain Structure of Periplasmic α-Amylase (MalS) from Glycoside Hydrolase Family 13 Subfamily 19.

Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120-180 (N-terminal) and 646-676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, ß-CD binds more weakly than the linear substrate, possibly due to the positioning of A402. MalS has two Ca2+ binding sites that contribute significantly to the thermostability of the enzyme. Intriguingly, the study found that MalS exhibits a high binding affinity for polysaccharides such as glycogen and amylopectin. The N domain, of which the electron density map was not observed, was predicted to be CBM69 by AlphaFold2 and might have a binding site for the polysaccharides. Structural analysis of MalS provides new insight into the structure-evolution relationship in GH13 subfamily 19 enzymes and a molecular basis for understanding the details of catalytic function and substrate binding of MalS.

A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase.

A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched ß-cyclodextrin (Glcn-ß-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (Km = 0.66 ± 0.02 mM and kcat = 76.7 ± 1.5 s(-1)) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis.

Reaction kinetics of substrate transglycosylation catalyzed by TreX of Sulfolobus solfataricus and effects on glycogen breakdown.

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-ß-cyclodextrin (Glc4-ß-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc4-ß-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-ß-cyclodextrin [(Glc4)2-ß-CD]; these were 6(1),6(3)- and 6(1),6(4)-dimaltotetraosyl-α-1,6-ß-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were 1.78 × 10(3) s(-1) and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10(3) s(-1) and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the kcat/Km ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.

Introducing transglycosylation activity in Bacillus licheniformis α-amylase by replacement of His235 with Glu.

To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM(-1)s(-1) for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM(-1)s(-1) for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235-located on a wide open groove near subsite +1-is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.

Mechanism of action of three different glycogen branching enzymes and their effect on bread quality.

Bread staling adversely affects the quality of bread, but starch modification by enzymes can counteract this phenomenon. Glycogen branching enzymes (GBEs) used in this study were isolated from Deinococcus geothermalis (DgGBE), Escherichia coli (EcGBE), and Vibrio vulnificus (VvGBE). These enzymes were characterized and applied for starch dough modification to determine their role in improving bread quality. First, the branching patterns, activity on amylose and amylopectin, and thermostability of the GBEs were determined and compared. EcGBE and DgGBE exhibited better thermostable characteristics than VvGBE, and all GBEs exhibited preferential catalysis of amylopectin over amylose but different degrees. VvGBE and DgGBE produced a large number of short branches. Three GBEs degraded the starch granules and generated soluble polysaccharides. Moreover, the maltose was increased in the starch slurry but most significantly in the DgGBE treatment. Degradation of the starch granules by GBEs enhanced the maltose generation of internal amylases. When used in the bread-making process, DgGBE and VvGBE increased the dough and bread volume by 9 % and 17 %, respectively. The crumb firmness and retrogradation of the bread were decreased and delayed significantly more in the DgGBE bread. Consequently, this study can contribute to understanding the detailed roles of GBEs in the baking process.

Association of novel domain in active site of archaic hyperthermophilic maltogenic amylase from Staphylothermus marinus.

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1-4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N' domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N' domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.

Isolation, Physicochemical Characterization, and Biological Properties of Inotodiol, the Potent Pharmaceutical Oxysterol from Chaga Mushroom.

Inotodiol, an oxysterol found only in Chaga mushroom, has received attention from the pharmaceutical industry due to its strong antioxidant and anti-allergic activities. However, the production of inotodiol is still challenging, and its fundamental properties have yet to be investigated. This study aims to develop an efficient method to produce high-purity inotodiol from Chaga mushroom. Then, pure inotodiol was used to assess its physicochemical properties and biological activities. By optimizing the solvent used for extraction and purification, a new method to produce inotodiol was developed with high purity (>97%) and purification yield (33.6%). Inotodiol exhibited a melting point (192.06 °C) much higher than lanosterol and cholesterol. However, the solubility of inotodiol in organic solvents was notably lower than those of the other two sterols. The difference in the hydroxyl group at C-22 of inotodiol has shown the distinctive physicochemical properties of inotodiol compared with cholesterol and lanosterol. Based on those findings, a nonionic surfactant-based delivery system for inotodiol was developed to improve its bioavailability. The inotodiol microemulsion prepared with 1-2% Tween-80 exhibited homogenous droplets with an acceptable diameter (354 to 217 nm) and encapsulation efficiency (85.6-86.9%). The pharmacokinetic analysis of inotodiol microemulsion in oral administration of 4.5 mg/kg exhibited AUC0-24h = 341.81 (ng·h/mL), and Cmax = 88.05 (ng/mL). Notably, when the dose increased from 4.5 to 8.0 mg/kg, the bioavailability of inotodiol decreased from 41.32% to 33.28%. In a mouse model of sepsis, the serum level of interleukin-6 significantly decreased, and the rectal temperature of mice was recovered in the inotodiol emulsion group, indicating that inotodiol microemulsion is an effective oral delivery method. These results could provide valuable information for applying inotodiol in functional food, cosmetic, and pharmaceutical industries.

One-step synthesis of glycogen-type polysaccharides from maltooctaose and its structural characteristics.

The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished based on the new findings of the catalytic mechanism of glycogen branching enzymes (GBEs) from Vibrio vulnificus, Deinococcus geothermalis, and Escherichia coli. GBEs from D. geothermalis and E. coli used maltononaose and maltotridecaose as the minimum, respectively, while V. vulnificus GBE (VvGBE) catalyzed the surprisingly small substrate, G8, into polysaccharides. Intriguingly, all three GBEs catalyzed α-1,4-transglycosylation at the early reaction stage of transglycosylation. VvGBE successfully converted the smallest substrate (G8) into two highly branched polysaccharides (HBP), in which the big polysaccharide (1.49 × 105 Da) exhibited structural properties similar to glycogen. Both HBPs had similar side chain distribution with a very short average degree of polymerization compared with mussel glycogen. As a molecular biology reagent, these nucleotide-free HBPs significantly increased the mRNA extraction efficiency of mammalian cells. Our results provide a new approach to the synthesis of novel polysaccharides.

Role of maltose enzymes in glycogen synthesis by Escherichia coli.

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.

A mesofluidic multiplex immunosensor for detection of circulating cytokeratin-positive cells in the blood of breast cancer patients.

We have recently reported the analytical performance of an immunosensor comprising one mm-scale parallel plate laminar flow chamber and applied to capture MCF7 breast cancer cells (Ehrhart et al., Biosens. Bioelectr. 24, 467, 2008). Herein we present a new multiplex immunosensor embodying four parallel plate laminar flow chambers that fit onto a standard, functionalized, microscopy glass slide. The four surfaces are coated with long alkyl chain spacers of 21-aminohenicosyl trichlorosilane (AHTS) and then grafted with a monoclonal anti-human epithelial cell adhesion molecule (EpCAM) antibody specific of target cells to immobilize. We first demonstrate a significantly (P < 0.01) improved capacity of each of the four flow chambers of the multiplex immunosensor to capture MCF7 cells compared to the previous single chamber device. Second, in addition to an increase of cell immobilization, the multiplex device offers a versatile tool easily grafted with various purified antibodies onto the four surfaces. Third, we obtained high cell capture rate and efficiency of various numbers of MCF7 cells spiked in buffer containing an equal number of background leukocytes. And fourth, we demonstrate isolation efficiency of circulating tumor cells (CTCs) from peripheral blood drawn from a small cohort of patients with localized or metastatic breast cancer. This new multiplex immunosensor could be tested for its potential to capture different subpopulations of CTCs.

Comparison of Catalyzing Properties of Bacterial 4-α-Glucanotransferases Focusing on Their Cyclizing Activity.

A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.

Structural and functional characteristics of clustered amylopectin produced by glycogen branching enzymes having different branching properties.

Three bacterial glycogen branching enzymes (GBEs) having different branching characteristics were used to produce clustered amylopectin (CAP), and structure and functional properties of CAPs were intensively analyzed. Branch distributions of three CAPs varied from very short (DPn = 6.65) to medium (DPn = 14.1). Branch distribution showed profound correlation with hydrodynamic diameter, water solubility, digestibility, and effects on mice gut-microbiota. All the CAPs showed nearly no viscosity and retrogradation. The very short-branch CAP exhibited more than 100-fold water-solubility, 3.5-fold lower α-amylase catalytic efficiency, and 27% lower digestibility in small intestine-mimicking condition than amylopectin. Intriguingly, medium branch CAP had 1.8-fold larger hydrodynamic diameter than the very short one. Mice gut-microbiota composition statistically varied after 12-day feeding of the CAPs, but only the medium chain CAP brought clear positive changes on the gut-microbiota. Consequently, slowly digestible starch was successfully synthesized by the single GBE, but the CAP structure affects in vivo functions in complicated manner.

Characterization of the Transglycosylation Reaction of 4-α-Glucanotransferase (MalQ) and Its Role in Glycogen Breakdown in Escherichia coli.

We first confirmed the involvement of MalQ (4-α-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ΔmalQ, showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-ß-CD: G4- ß-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.

Characterization and thermal inactivation kinetics of highly thermostable ramie leaf ß-amylase.

We characterized ramie leaf ß-amylase, and determined its thermostability and kinetic parameters. The enzyme was purified 53-fold using ammonium sulfate fractionation (40-60% saturation), anion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Superdex-200. The purified enzyme was identified as ß-amylase with molecular mass of 42kD. The enzyme displayed Km and kcat values for soluble potato starch of 1.1mg/mL and 7.8s-1, respectively. The enzyme had a temperature optimum of 65°C, and its activity at 70°C was 92% of that at the optimal temperature after a 15-min incubation. Furthermore, enzyme activity was stable during treatment at 55°C for 60min but was inactivated rapidly at >75°C. This thermal behavior indicates that ramie leaf ß-amylase has excellent intermediate temperature-stable enzyme properties for the baking and bio-industries. Inactivation of the enzyme followed first-order kinetics in the range of 55-80°C. The enthalpy change of thermal inactivation (ΔH‡), ΔG‡, and ΔS‡ were 237.2kJ/mol, 107.7kJ/mol, and 0.39kJ/molK at 333K, respectively. The D-value at 65°C (=110min) and the z-value (=9.4°C) are given for food processing.

Gene therapy for hepatocellular carcinoma using non-viral vectors composed of bis guanidinium-tren-cholesterol and plasmids encoding the tissue inhibitors of metalloproteinases TIMP-2 and TIMP-3.

Metalloproteinases (MMPs) and their natural inhibitors (TIMPs) contribute to the regulation of tumor microenvironment. Their expressions are deregulated in almost all human cancers. We report a novel approach to gene therapy of hepatocellular carcinoma (HCC), using repeated injections of DNA plasmids encoding the tissue inhibitors of metalloproteinases (TIMPs) TIMP-2 or TIMP-3, and a novel competent formulation of gene transfer based on nontoxic cationic cholesterol derivatives. The new gene delivery system was efficient in demonstrating the antitumor efficiency of TIMP-2 or TIMP-3 in inhibiting tumor growth of human HuH7 HCC cells xenografted into nude mice. We show, for the first time, an in vivo effect of TIMP-3 in delaying HCC tumor growth. No treatment-related toxicity was noted. An inhibition of angiogenesis and tumor necrosis accompanied the inhibitory effects of TIMP-2 or TIMP-3 on tumor expansion and invasion. We also report a bystander effect produced by transfected HuH7 tumor cells mixed with untransfected cells in 1:1 ratio in culture that resulted in killing 98% of cells within 96 h. In addition, the soluble forms of TIMP-2 and TIMP-3 expressed by transfected cells exerted a cytotoxic effect on untransfected HuH7 cell cultures. Taken together, these results demonstrate the potential efficacy of repeated treatment of secreted TIMP-2 and TIMP-3 for the design of nonviral gene therapy for hepatocarcinoma.

Experimental evidence for a 9-binding subsite of Bacillus licheniformis thermostable α-amylase.

The action pattern of Bacillus licheniformis thermostable α-amylase (BLA) was analyzed using a series of (14)C-labeled and non-labeled maltooligosaccharides from maltose (G2) to maltododecaose (G12). Maltononaose (G9) was the preferred substrate, and yielded the smallest Km=0.36 mM, the highest kcat=12.86 s(-1), and a kcat/Km value of 35.72 s(-1) mM(-1), producing maltotriose (G3) and maltohexaose (G6) as the major product pair. Maltooctaose (G8) was hydrolyzed into two pairs of products: G3 and maltopentaose (G5), and G2 and G6 with cleavage frequencies of 0.45 and 0.30, respectively. Therefore, we propose a model with nine subsites: six in the terminal non-reducing end-binding site and three at the reducing end-binding site in the binding region of BLA.

Characterization and application of an acidophilic and thermostable ß-glucosidase from Thermofilum pendens.

The gene encoding a ß-glucosidase from the archaeon Thermofilum pendens (Tpbgl) was cloned and expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 77.8 kDa and released glucose or mannose from p-nitrophenyl-ß-d-glucopyranoside (pNPG), cellobiose, mannobiose, and genistin. Peak Tpbgl activity was detected at 90°C, and 50% activity remained after incubation for 60 min at 95°C. The optimal pH for pNPG hydrolysis was 3.5. When the enzyme was incubated with pNPG in the presence of ethanol and propanol, the glucose moiety was transferred to acceptor alcohols. Tpbgl is the archaeal ß-glucosidase from glucoside hydrolase family 3 and found to be most heat stable under extremely acidic conditions (pH 3.5). The kinetic parameters revealed that Tpbgl had the highest catalytic efficiency toward pNPG (kcat/Km = 3.05) with strong substrate affinity for such natural substrates as cellobiose (Km = 0.149) and mannobiose (Km = 0.147). Genistin solubilized in 10-40% DMSO was hydrolyzed to genistein with nearly 99% conversion, indicating that high concentrations of the water-insoluble isoflavone glycoside can be treated by the enzyme. Our results indicate that Tpbgl has great potential in cellulose saccharification and the glucoside hydrolysis of natural compounds.

A new immunosensor for breast cancer cell detection using antibody-coated long alkylsilane self-assembled monolayers in a parallel plate flow chamber.

We designed a new efficient and reliable immunosensor and demonstrated its analytic performance to capture breast cancer MCF7 and T47D cells, under laminar flow, onto antibody-coated long alkylsilane self-assembled monolayers (SAMs) in a parallel plate flow chamber. The surface floor of the laminar flow chamber was grafted with an amino-terminated long alkyl chain spacer, 21-aminohenicosyl trichlorosilane (AHTS) followed by tethering a specific monoclonal antibody directed against the human epithelial cell adhesion molecule (EpCAM) antigen, which is overexpressed in primary breast cancer. Properties of the AHTS- and antibody-grafted surface floor were compared to that of surface floors coated with the short alkyl spacers 3-glycidoxy-propyl trimethoxysilane (GPTS) or 3-aminopropyl triethoxysilane (APTES) and antibodies. A theoretical model was constructed according to the geometry of the flow chamber in order to calculate the trajectories that would use cell flows. Cell capture experiments demonstrated that cell immobilization was optimized throughout the whole flow chamber. High cell capture was yielded on antibody-tethered long alkyl AHTS surface. This new procedure offers multiple advantages: a versatile tool readily applied to a panel of purified antibodies, an enrichment of cell immobilization using repetitive cell flow, and a stable capturing surface suitable for long term storage and handling.

Protocadherin-PC promotes androgen-independent prostate cancer cell growth.

BACKGROUND: Protocadherin-PC (PCDH-PC) expression is upregulated in apoptosis-resistant sublines of the LNCaP human prostate cancer (CaP) cell line. Here, we assess the role of PCDH-PC in CaP cells and its mRNA expression in human prostate tissues. METHODS: LNCaP cells transfected with PCDH-PC were tested for their ability to grow in vitro and in vivo in androgen-deprived conditions. PCDH-PC mRNA expression was evaluated by semi-quantitative RT-PCR and by in situ hybridization. RESULTS: PCDH-PC expression induced Wnt signaling in CaP cells and permitted androgen-independent growth of hormone-sensitive CaP cells. Expression of PCDH-PC-homologous transcripts was low and restricted to some epithelial cells in normal tissue and to CaP cells in tumors. However, hormone-resistant CaP cells expressed significantly higher levels of PCDH-PC-related mRNA. CONCLUSIONS: Our findings suggest a novel mechanism for the progression of CaP involving expression of PCDH-PC. This novel protocadherin induces Wnt signaling, promotes malignant behavior and hormone-resistance of CaP cells.

Inhibition of rat hepatocellular carcinoma tumor growth after multiple infusions of recombinant Ad.AFPtk followed by ganciclovir treatment.

BACKGROUND/AIMS: The antitumor efficiency of thymidine kinase (tk) in Herpes Simplex virus-tk-based gene therapy of rat hepatocellular carcinoma (HCC) was examined by specific transcriptional targeting of tk to tumor cells by the alpha-fetoprotein (AFP) gene promoter and by multiple infusions of recombinant adenovirus Ad.AFPtk. METHODS: We developed a surgical procedure that allows efficient, non-invasive delivery (during 2 months) of recombinant Ad via the intra-hepatic artery (IHA) route. RESULTS: Treatment of tumor-bearing rats with either three or five doses of 5x10(9)pfu Ad.AFPtk, administered every 3 days, and followed by intra-peritoneal treatment with ganciclovir (GCV), resulted in tumor growth inhibition and apoptosis, when compared to untreated tumor-bearing rats or animals treated with Ad.AFPlacZ or buffered saline. No treatment-related toxicity was noted. Antitumor efficacy, based on tumor size and number of tumors, was demonstrated in more than 50% of Ad.AFPtk+GCV-treated rats, as compared to control rats (P<0.0005). CONCLUSIONS: Our results demonstrate the safety and potential of multiple Ad.AFPtk administrations by the IHA route to inhibit HCC tumor growth, and support further clinical investigation of Ad.AFPtk gene therapy for treatment of multifocal tumor lesions in most primary liver cancers.