Gel Size Exclusion Chromatography Research Articles

Long-term shelf-life liposomes for delivery of prednisolone and budesonide

Liposomes are nanoscale drug delivery systems built up from lipid layers and are able to spontaneously self-assemble in an aqueous environment. Both hydrophilic and hydrophobic drugs can be delivered by liposomes and this kind of nanoformulation offers many advantages regarding biodistribution, drug absorption and controlled drug release. Corticosteroids as lipophilic molecules are able to integrate into the lipid bilayer. This novel approach can improve the efficacy of several anti-inflammatory, such as asthma therapy.Our aim was to create liposomes with long shelf-life, which can incorporate and release corticosteroids such as Prednisolone (Pred) and Budesonide (Bud) at the temperature of inflamed tissues. Two kinds of liposome samples were prepared from three different kinds of phospholipids to get unilamellar vesicles with 100 nm in diameter and characterize their physicochemical properties and effect on living cells. Their main phase transition temperature in the physiologically relevant temperature range was measured by differential scanning calorimetry. According to the size distributions determined by dynamic light scattering, all drug-containing liposomes were stable for 6 months. All of the liposome types have a slightly negative zeta potential value. The Fourier-transform infrared spectroscopy revealed no chemical interaction between the drug and lipid molecules. The entrapment efficacy was determined by size-exclusion gel chromatography combined with UV–VIS spectrophotometry and it was very high in both cases (between 70 and 87%). The drug leakage was 35–40% for Pred and 6–8% for Bud in the first 30 min. The effect of liposomal drugs on cell viability was measured on the EBC-1 human lung carcinoma cell line. Neither the free corticosteroids nor their liposomal form were toxic to the cells. The cellular internalization of the liposomes was proved by flow cytometry and confocal microscopy.In summary, these liposomes could be useful in the delivery of corticosteroids (Pred or particularly Bud) in more effective asthma therapy, having fewer side effects due to the nanoformulation.

Altered properties of amyloidogenic prion protein in genetic Creutzfeldt-Jakob disease with PRNP V180I mutation in response to pentosan polysulfate.

Genetic Creutzfeldt-Jakob disease (gCJD) with V180I prion protein gene (PRNP) mutation shows weaker prion protein (PrP) deposition histologically compared with sporadic CJD, and it is more difficult to detect protease-resistant prion protein in immunoblotting. However, we previously reported the autopsy case of a patient with V180I gCJD who was treated with pentosan polysulfate sodium (PPS); this case had increased protease-resistant PrP deposition. It has been suggested that PPS might reduce protease-resistant PrP; however, the detailed pharmacological and histopathological effects of PPS in humans remain unknown. We examined autopsied human brain tissue from four cases with V180I gCJD that were added to our archives between 2011 and 2021: two cases treated with PPS and two cases without PPS. We conducted a neuropathological assessment, including immunohistochemistry for PrP. We also performed immunoblotting for PrP on homogenate samples from each brain to detect protease-resistant PrP using both a conventional procedure and size-exclusion gel chromatography for the purification of oligomeric PrP. Both PPS-treated cases showed long survival time over 5 years from onset and increased PrP deposition with a characteristic pattern of coarse granular depositions and congophilic PrP microspheres, whereas the cases without PPS showed around 1-year survival from onset and relatively mild neuronal loss and synaptic PrP deposition. Although cortical gliosis seemed similar among all cases, aquaporin 4-expression as a hallmark of astrocytic function was increased predominantly in PPS cases. Immunoblotting of non-PPS cases revealed protease-resistant PrP in the oligomeric fraction only, whereas the PPS-treated cases showed clear signals using conventional procedures and in the oligomeric fraction. These unique biochemical and histopathological changes may reflect the progression of V180I gCJD and its modification by PPS, suggesting the possible existence of toxic PrP-oligomer in the pathophysiology of V180I gCJD and beneficial effects of PPS toward the aggregation and detoxication of toxic PrP-oligomer.

Physicochemical parameters optimization and peroxidase characterization from Aspergillus niger native strain by solid-state fermentation for improved dye decolorization

The hyper yield of peroxidase (POX) was investigated for a novel native Aspergillus niger strain identified by 18S RNA analysis. A. niger strains sequences were submitted to GenBank; IDs allotted were MN611114.1 (BMB 17) and MN559756.1 (BMB-18). The identified Aspergillus strains in combination showed enhanced (POX) activity (601.5 U/mL) by solid-state fermentation in comparison to their individual activities. POX was purified by ammonium sulfate, and size exclusion gel chromatography exhibited a 7.83-fold increase in POX concentration (13.3 U/mg) in comparison to BMB17 and BMB 18 (11.8 & 7.6 U/mg respectively). The best POX activity was obtained with pH 6.5, 37 °C, and 5 days of incubation. Using guaiacol as substrate, POX showed maximum activity (Vmax) of 537 U/mL with a corresponding Michaelis constant (Km) value of 126 µM. Calcium chloride worked as a POX activator at 300 & 400 mM. Zinc sulfate (500 mM), EDTA (5 mM); ethanol, propanol, and acetonitrile (50%) inhibited (18-30%) POX. Urea (1M), and copper sulfate (500 mM) strongly inhibited POX up to 40%. Polysorbate-80 (1%) slightly reduced the POX by 10% to 15%. BMB17+18-induced promising dye decolorization (88-98%) against all vat dyes, methylene blue, and phenol red.

Pathogenic antibody response to glucose-6-phosphate isomerase targets a modified epitope uniquely exposed on joint cartilage

ObjectivesTo identify the arthritogenic B cell epitopes of glucose-6-phosphate isomerase (GPI) and their association with rheumatoid arthritis (RA).MethodsIgG response towards a library of GPI peptides in patients with early RA,...

Comprehensive application possibility: Construction hydrophilic, amphiphilic and hydrophobic system of modified zein by enzymatic or cysteine modification

Zein, a hydrophobic protein due to high percentage of apolar amino acids, which limits its modification and wide application in the food industry. The interaction between the molecule and water will be changed by introducing different hydrophilic groups into zein, which is the basis aimed at food application. Therefore, zein was modified based on construction of zein-potassium oleate system to obtain hydrophilic, amphiphilic and hydrophobic systems, thus the structure and properties of modified zein were explored in the present study. The results of the three-phase contact angle showed that the constructed zein-potassium oleate system is a hydrophobic system. After enzymatic modification, a hydrophilic system can be obtained. Amphiphilic system can be obtained by cysteine modification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, gel size exclusion chromatography, hydrophilic-lipophilic balance value, free amino group and sulfhydryl group results can also prove the hydrophilic and hydrophobic system obtained by different modification methods. Ultraviolet, Infrared and fluorescence spectra of zein after enzymatic hydrolysis and cysteine modification indicated that the hydrophilicity and hydrophobicity of the zein microenvironment also changed. This study may provide a feasible approach to broaden the application of zein in food industry. • Two modification methods were used to realize the construction of different systems of zein. • The formation of different systems is directly or indirectly demonstrated in a number of ways. • This paper provides a feasible approach to broaden the application of zein in food industry.

Enzymatic Pea Protein Hydrolysates Are Active Trypsin and Chymotrypsin Inhibitors.

In this work, we report the potency of enzymatic hydrolysates of pea proteins against trypsin and chymotrypsin. Pea protein concentrate was digested with each of alcalase, chymotrypsin, pepsin, and trypsin, followed by membrane separation of the protein hydrolysates into peptide fractions (<1, 1–3, 3–5, and 5–10 kDa). Peptide size profiling with size-exclusion gel chromatography indicated the narrowest size range (0.85–4.98 kDa) for alcalase. Trypsin activity was strongly (p < 0.05) inhibited by the ultrafiltration fractions (mean IC50 = 2.2 mg/mL) obtained from the trypsin hydrolysate when compared to the unfractionated hydrolysate (IC50 = 6.8 mg/mL). Similarly, ultrafiltration also enhanced trypsin inhibition by the alcalase-digested peptides with an IC50 of 21.4 mg/mL for the unfractionated hydrolysate in comparison to 3.1–4.7 mg/mL for the fractions. However, ultrafiltration did not enhance trypsin inhibitory activity of chymotrypsin-digested peptides, while the peptide separation reduced efficacy of pepsin-digested peptides. In contrast, chymotrypsin inhibition by all the enzymatic digests was significantly (p < 0.05) enhanced by ultrafiltration, especially peptide sizes >3 kDa. Kinetics of enzyme inhibition indicate peptides were bound to the enzyme active site in a competitive mode that led to reduced catalysis. We conclude that the pea peptides could function as useful tools to promote human health and as a preservative during food processing and storage.

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in ‘sub G0/G1’ phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.

Abstract 566: MicroRNA-146a Deficiency Prevents PCSK9 Gain-of-Function Mutation-induced Hypercholesterolemia in Mice

Background and Objective: Mimetic mediated activation of microRNA 146a (miR-146a) reduces atherosclerosis via suppression of nuclear factor-κB-driven inflammation in mice. The purpose of this study was to determine whether miR-146a influences plasma cholesterol in hypercholesterolemic mice. Methods and Results: To induce hypercholesterolemia, female C57BL/6 miR-146a WT (n=8) and miR-146a KO (n=8) mice were injected intraperitoneally with an adeno-associated viral vector (AAV) expressing the proprotein convertase subtilisin/kexin type 9 (PSCK9 D377Y) gain-of-function mutant at a dose of 3 x 10 10 genomic copies/mouse. After infection, mice were fed a Western diet (21% wt/wt milk fat; 0.15% wt/wt cholesterol) for sixteen weeks, and plasma PCSK9 and total cholesterol concentrations were monitored monthly using an enzymatic assay. Plasma PCSK9 concentrations were profoundly increased 4 weeks post injection (Baseline: WT - 179 ± 12 vs KO - 207 ± 12; Week 4: WT - 1700 ± 148 vs KO - 2689 ± 305 ng/ml) and remained significantly high during 16 weeks (WT - 882 ± 142 vs KO - 718 ± 109 ng/ml; p&lt;0.05 vs baseline) of Western diet feeding. Consistent with increased plasma PCSK9 concentrations, plasma cholesterol concentrations were increased in both groups of mice. Interestingly, miR-146a KO group mice showed less significant increase in plasma cholesterol compared to WT group (Baseline: WT - 88 ± 3 vs KO - 83 ± 3; Week 4: WT - 328 ± 25 vs KO - 195 ± 18 mg/dl) irrespective of the comparable plasma PCSK9 concentrations. Also, lipoprotein distribution analysis with size exclusion gel chromatography revealed that miR-146a KO mice showed a strong reduction in high density lipoprotein (HDL) particles while very low density lipoprotein (VLDL) and low density lipoprotein (LDL) particles were not affected. Conclusion: Our findings suggests that miR146a plays a critical role in the regulation of HDL particles in PCSK9 gain-of-function mutant-induced hypercholesterolemia in mice. Future studies will identify gene targets influenced by miR-146a in regulating HDL-cholesterol in hypercholesterolemic mice.

Effect of single-chirality single-walled carbon nanotubes in dye sensitized solar cells photoanodes

Dye-sensitized solar cells (DSSCs) with photoanodes composed of chirality selected single-wall carbon nanotubes (SWNTs) have been fabricated and tested for first time. Single chirality SWCNTs separation (93% purity) have been achieved by modifying standard size exclusion gel chromatography. Chirality selection has allowed for “tuning” of the energy barrier at the TiO2/SWNT/FTO interface, electronic conductivity enhancement, and reduced SWCNTs-ruthenium dye competition for light absorption resulting in a 81% energy conversion efficiency improvement compared to mixed chirality cells. Unfortunately, energy conversion efficiency has been limited by cells’ low shunt resistance. Additionally, SWCNTs electron transfer properties have been exploited to prepare mixed and chirality specific CNTs based DSSCs’ counter electrodes.

Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells

The objective of this study was to investigate the intracellular reactive oxygen species (ROS) scavenging activities and antioxidant enzyme regulating capacities of corn gluten peptide fractions (CPFs) in HepG2 cells. A cellular antioxidant activity (CAA) assay was used to assess their antioxidant activities and revealed that both CPF1 (molecular weight <1kDa) and CPF2 (molecular weight between 1 and 3kDa) exhibited high cellular antioxidant activities with EC50 values of 2.85±0.19mg/mL and 5.05±0.32mg/mL, respectively. Both CPFs also exhibited cytoprotective effects and intracellular ROS scavenging activities in HepG2 cells subjected to oxidative stress by oxidation with H2O2. In addition, at concentrations of 2.50mg/mL, the CPFs increased the activity levels of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), as well as the total glutathione (GSH) levels in oxidized HepG2 cells (from 86.54% to 114.14% (CPF1) or 109.72% (CPF2) for SOD activity; from 71.91% to 107.64% (CPF1) or 106.50% (CPF2) for CAT activity; from 70.52% to 103.01% (CPF1) or 104.10% (CPF2) for GR activity; and from 81.39% to 114.00% (CPF1) or 108.82% (CPF2) for total GSH levels). These results suggested that both CPF1 and CPF2 exhibited positive effects on the activities of the intracellular antioxidant enzymes SOD, CAT and GR, as well as on the total GSH levels in HepG2 cells under conditions of oxidative stress. Furthermore, size exclusion gel chromatography and MALDI-TOF/TOF mass spectrometry revealed that the molecular weights of the antioxidant peptides in CPF1 were between 500Da to 900Da, and a novel antioxidant peptide consisting of GLLLPH (Gly-Leu-Leu-Leu-Pro-His) was identified in CPF1.

Relationship of adiponectin and its multimers to metabolic indices in cats during weight change

Adiponectin is an important anti-inflammatory hormone secreted from adipose tissue. The high-molecular-weight form of adiponectin (HMW) closely correlates with insulin sensitivity in human beings. This study uses a novel method of size-exclusion gel chromatography combined with enzyme-linked immunosorbent assay to measure HMW feline adiponectin and determine its relationship to leptin, cholesterol, and insulin sensitivity as cats gain and lose weight. In addition, total adiponectin and its messenger RNA expression in subcutaneous adipose tissue were measured. No correlations were found between total serum adiponectin and subcutaneous adipose messenger RNA expression, fat mass, or measures of insulin sensitivity. This study demonstrates that cats have high percentages of HMW adiponectin. Although weak correlations between HMW adiponectin and fat mass were detected, additional cats are needed to determine if the correlations are significant.

Abstract 293: ApoB-Containing Lipoproteins Contribute to Augmentation of AngII-Induced Abdominal Aortic Aneurysms

Background and objective: Presence of abdominal aortic aneurysms (AAA) is associated with dyslipidemia in humans. Hypercholesterolemic mice having a predominance of ApoB-containing lipoproteins are more susceptible to Angiotensin (Ang)II-induced AAA formation. A recent publication demonstrated that exogenously delivered high-density lipoprotein (HDL) prevented AngII-induced AAAs. ApoAI is the major apolipoprotein of HDL. The purpose of this study was to determine whether manipulations of ApoAI or ApoB-containing lipoproteins influence the development of AngII-induced AAA. Methods and Results: Eight week old male ApoAI +/+ or -/- mice with either wild type or LDL receptor -/- background were fed normal diet and infused with AngII (1 mg/kg/min) for 28 days. Plasma lipoprotein distributions were resolved by size exclusion gel chromatography. Deficiency of ApoAI led to profound reductions of plasma HDL-C concentrations in both strains. However, ApoAI deficiency did not exacerbate AngII-induced AAA as determined by maximal outer widths of suprarenal aortas (ApoAI +/+ versus -/-:1.52 ± 0.25 versus 1.22 ± 0.18 mm, P= 0.2 for C57BL/6 strain;1.21 ± 0.18 versus 1.11 ± 0.17 mm, P= 0.2 for LDLR -/- strain). Ezetimibe, an intestinal cholesterol absorption inhibitor, was administered to eight week old male ApoE -/- mice fed normal diet. Ezetimibe decreased plasma cholesterol concentrations from 451 ± 8 to 204 ± 18 mg/dl (P&lt;0.001). This reduction was due to reductions in ApoB-containing lipoproteins. Administration of ezetimibe decreased AngII-induced AAA (control versus ezetimibe: 2.25 ± 0.28 mm versus 1.37 ± 0.1 mm, P=0.009). The same dose of ezetimibe was also administered to ApoE -/- mice fed western diet. Plasma cholesterol concentrations were reduced (from 980 ± 86 to 546 ± 11 mg/dl, P&lt;0.001) attributed to reductions of apoB-containing lipoproteins. Administration of ezetimibe led to a profound reduction of atherosclerosis in western diet fed apoE -/- mice, but did not attenuate AngII-induced AAAs. Conclusion: ApoB-containing lipoproteins, rather than HDL, contribute to the augmentation of AngII-induced AAAs. However, substantial reductions in apoB-containing lipoproteins were required to attenuate AngII-induced AAA.

Comparative Study About Some Physical Properties, In vitro Digestibility and Immunoreactivity of Soybean Protein Isolate for Infant Formula

The objective of this study was to determine molecular weight subunit distributions of soy protein isolate (SPI) by SDS-PAGE and gel size exclusion chromatography, and further to investigate the differences of thixotropy, viscosity, in vitro digestibility and immunoreactivity of SPI for infant formula produced in Chinese domestic companies and world famous SPI manufacturers such as Dupont and Fuji. The molecular subunit distributions were analyzed by SDS-PAGE and gel size exclusion chromatography, indicating that Solae and Fuji were hydrolyzed by proteolytic enzymes. The thixotropy of Fuji and Solae significantly reduced around by 95 %, compared with those of Mantianxue and Dupont Zhengzhou. The allergen contents of Fuji and Solae strikingly decreased by 60 and 84 % respectively, in contrast to that of Mantianxue. The in vitro protein digestibility of Solae at the end of pepsin and trypsin digestion markedly increased by 11.7 and 11.3 %, respectively, in comparison to those of Mantianxue. Suitable enzymatic hydrolyzed SPIs showed lower thixotropy, viscosity, immunoreactivity and higher in vitro protein digestibility than those from the other SPIs. The lower thixotropy indicates low difficulty or shear stress in swallowing for infants. The lower immunoreactivity will improve the safety of SPI for cow milk allergic babies.

Abstract 285: The Effects of Hypercholesterolemia and Lipoprotein-Cholesterol Concentrations on Angiotensin II-Induced Abdominal Aortic Aneurysms

Background and objective Angiotensin II (AngII) infusion promotes abdominal aortic aneurysms (AAA) in both normo- and hypercholesterolemic mice, but the latter have a greater susceptibility, implicating a role of hypercholesterolemia in AngII-induced AAA. A recent publication demonstrated that high-density lipoprotein (HDL) administration prevented AngII-induced AAA in apoE -/- mice without affecting total plasma cholesterol concentrations. The purpose of this study was to determine the relationship between concentrations and characteristics of plasma cholesterol and lipoproteins and AngII-induced AAA. Methods and Results Three strains of male mice were studied: C57BL/6, LDL receptor -/-, and apoE -/-. These mice were fed either a normal diet or a saturated fat- enriched diet. AngII (1 μg/kg/min) was infused for 4 weeks through osmotic minipumps implanted subcutaneously. Total plasma cholesterol concentrations were measured using an enzymatic kit. Lipoproteins were resolved by size exclusion gel chromatography and non-HDL and HDL analyzed using Peakfit software. AAA was quantified by measuring maximal outer width of suprarenal aortas. In C57BL/6 mice, the fat-enriched diet modestly increased concentrations of total plasma cholesterol (114 ± 4 vs 99 ± 6 mg/dl; P=0.03) and HDL-C (98 ± 4 vs 81 ± 7 mg/dl; P=0.02), while did not change non-HDL-C/HDL-C ratio (0.21 ± 0.03 vs 0.24 ± 0.03; P=0.5) and AAA (1.1 ± 0.1 vs 1.1 ± 0.1 mm; P=0.6). In LDL receptor -/- mice, the fat-enriched diet feeding increased concentrations of total plasma cholesterol (1590 ± 58 vs 276 ± 9 mg/dl; P&lt; 0.001), HDL-C (132 ± 5 vs 90 ± 3 mg/dl; P&lt;0.001), non-HDL-C/HDL-C ratio (10.8 ± 0.3 vs 2.1 ± 0.1; P&lt;0.001), and AAA (1.6 ± 0.1 vs 1.2 ± 0.1 mm; P=0.002), compared to LDL receptor -/- mice fed the normal diet. In apoE -/- mice, the fat-enriched diet increased concentrations of total plasma cholesterol (1261 ± 121 vs 348 ± 38 mg/dl; P&lt;0.001), had no effect on HDL-C, increased non-HDL-C/HDL-C ratio (53.1 ± 5.9 vs 10.5 ± 2.0; P=0.016), and had no effect on AAA development (1.5 ± 0.2 vs 1.6 ± 0.2 mm; P=0.4). Conclusion Hypercholesterolemia promotes AngII-induced AAA, while AAA has no clear relationship with either concentrations of total plasma cholesterol or HDL-C.

Protease‐resistant PrP and PrP oligomers in the brain in human prion diseases after intraventricular pentosan polysulfate infusion

Intraventricular infusion of pentosan polysulfate (PPS) as a treatment for various human prion diseases has been applied in Japan. To evaluate the influence of PPS treatment we performed pathological examination and biochemical analyses of PrP molecules in autopsied brains treated with PPS (one case of sporadic Creutzfeldt-Jakob disease (sCJD, case 1), two cases of dura mater graft-associated CJD (dCJD, cases 2 and 4), and one case of Gerstmann-Sträussler-Scheinker disease (GSS, case 3). Six cases of sCJD without PPS treatment were examined for comparison. Protease-resistant PrP (PrP(res) ) in the frontal lobe was evaluated by Western blotting after proteinase K digestion. Further, the degree of polymerization of PrP molecules was examined by the size-exclusion gel chromatography assay. PPS infusions were started 3-10 months after disease onset, but the treatment did not achieve any clinical improvements. Postmortem examinations of the treated cases revealed symmetrical brain lesions, including neuronal loss, spongiform change and gliosis. Noteworthy was GFAP in the cortical astrocytes reduced in all treated cases despite astrogliosis. Immunohistochemistry for PrP revealed abnormal synaptic deposits in all treated cases and further plaque-type PrP deposition in case 3 of GSS and case 4 of dCJD. Western blotting showed relatively low ratios of PrP(res) in case 2 of dCJD and case 3 of GSS, while in the treated sCJD (case 1), the ratio of PrP(res) was comparable with untreated cases. The indices of oligomeric PrP were reduced in one sCJD (case 1) and one dCJD (case 2). Although intraventricular PPS infusion might modify the accumulation of PrP oligomers in the brains of patients with prion diseases, the therapeutic effects are still uncertain.

Conjugal plasmid transfer inStreptomycesresembles bacterial chromosome segregation by FtsK/SpoIIIE

Conjugation is a major route of horizontal gene transfer, the driving force in the evolution of bacterial genomes. Antibiotic producing soil bacteria of the genus Streptomyces transfer DNA in a unique process involving a single plasmid-encoded protein TraB and a double-stranded DNA molecule. However, the molecular function of TraB in directing DNA transfer from a donor into a recipient cell is unknown. Here, we show that TraB constitutes a novel conjugation system that is clearly distinguished from DNA transfer by a type IV secretion system. We demonstrate that TraB specifically recognizes and binds to repeated 8 bp motifs on the conjugative plasmid. The specific DNA recognition is mediated by helix α3 of the C-terminal winged-helix-turn-helix domain of TraB. We show that TraB assembles to a hexameric ring structure with a central ∼3.1 nm channel and forms pores in lipid bilayers. Structure, sequence similarity and DNA binding characteristics of TraB indicate that TraB is derived from an FtsK-like ancestor protein, suggesting that Streptomyces adapted the FtsK/SpoIIIE chromosome segregation system to transfer DNA between two distinct Streptomyces cells.

Evolutionary conservation of mannan-binding lectin (MBL) in bony fish: Identification, characterization and expression analysis of three bona fide collectin homologues of MBL in the rainbow trout ( Onchorhynchus mykiss)

The complement system of fish is generally as complex as in mammals, and in addition Teleost fish often possess several genes encoding different subtypes of a given complement component, such as C3-1, C3-3 and C3-4. Initiators of both the classical (C1) and alternative pathway (factor B) have been characterized in the rainbow trout but so far no molecules of the lectin pathway have been identified. Based on the generally accepted idea of complement evolution, which predicts that the alternative pathway predates the two other pathways, and that the lectin pathway developed before the classical, we set out to characterize members of the lectin pathway in fish. We identified and characterized three homologues of mannan-binding lectin (MBL) with a bona fide collectin structure. By means of RT-PCR and immunohistochemistry using monoclonal antibodies we found that they were synthesized in the spleen, the anterior intestine and the liver. In the liver, we saw co-expression with mannan-binding lectin associated serine protease (MASP). The MBL homologues 2 and 3 (MBL-H2,3) were also found in the vascular system of the rainbow trout. By means of gel size exclusion chromatography of serum we found that MBL-H2,3 oligomerized heterogeneously from monomers to tetramers of a trimeric collagenous subunit. Sequence comparison and phylogenetic studies showed that the homologues were more related with MBL than any other collectins, and that two previously characterized trout proteins, designated MBL1 and MBL2, should be reconsidered as MBL candidates.

Construction of non-covalent single-chain Fv dimers for hepatocellular carcinoma and their biological functions

To create new diabodies with improved binding activity to antigen of the variable light - variable heavy (VH-VL) oriented single-chain Fv dimers genes (scFv). The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E. The recombinant plasmids were transformed into TG1 cells and sequenced. The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies. Expressed products were identified by SDS-PAGE, Western blotting, size exclusion gel chromatography (SEC), ELISA and immunohistochemistry. Three scFv (scFv-3, scFv-4, scFv-5) were constructed successfully with binding ability to hepatocellular carcinoma 3.5-6 fold greater than their parental scFv. The single-chain Fv dimer (scFv-5, termed BDM3) with the best binding ability was successfully expressed in Yeast pichlia, as shown by. SDS-PAGE and Western blotting. SEC results suggested the molecular weight of the expressed products was about 61 kDa. Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv, still having 50% binding activity even after 16 h incubation as 37°C. The purified dimers were bound specifically to the tumor antigen of HCC. we have generated scFv dimers by shortening a series of linkers to 3-5 amino acid residues in VH-linker-VL orientation, resulting in highly stable and affinity-improved dimeric molecules. These will become an attractive targeting moiety in immunotherapeutic and diagnostic applications for HCC.

Metastasis-inducing S100A4 protein is associated with the disease activity of rheumatoid arthritis

To evaluate the association between metastasis-inducing protein S100A4 and disease activity in patients with RA, and to demonstrate the effect of TNF-alpha blocking therapy on plasma levels of S100A4 in these patients. Plasma levels of the S100A4 protein were analysed in 40 anti-TNF-alpha naive patients with active RA. Of the 40 patients, 25 were treated with adalimumab and monitored over time. The conformational form of S100A4 was analysed using size-exclusion gel chromatography. TNF-alpha mRNA expression and protein synthesis were analysed by RT-PCR and ELISA, respectively. Baseline levels of S100A4 were significantly correlated with disease activity in RA patients (r = 0.41; P < 0.01). After 12 weeks of treatment with adalimumab, there was an obvious shift in the conformations of S100A4 from the multimeric to the dimeric forms, whereas the total levels of the S100A4 protein remained unchanged. This suggests that the bioactive (multimer) S100A4 may decline in response to successful treatment with adalimumab. In addition, we showed significant up-regulation of TNF-alpha mRNA (P < 0.01), and protein release to the cell culture medium of monocytes stimulated with the S100A4 multimer compared with those treated with the dimer and to the unstimulated monocytes (P < 0.001). This is the first study to show that the levels of the S100A4 protein are correlated with RA disease activity. Furthermore, only the bioactive form, but not the total amount of S100A4, decreases after successful TNF-alpha blocking therapy in patients with RA. These data support an important role for the S100A4 multimer in the pathogenesis of RA.

Development of oligomeric prion‐protein aggregates in a mouse model of prion disease

In prion diseases the normal cellular isoform of prion protein (PrP), denoted PrP(C), is converted into an abnormal, pathogenic isoform of PrP (PrP(Sc)). Diagnostic tools for prion diseases are conventionally based on the detection of protease-resistant PrP (PrP(res)) after proteinase K digestion. However, recent studies have revealed that protease-sensitive abnormal PrP (sPrP(Sc)) also exists in significant amounts in brains suffering from prion diseases. Here, we designed a simplified size-exclusion gel chromatography assay, using disposable spin columns to examine PrP aggregates in the course of the disease, without proteinase K digestion. Brain homogenates of NZW mice, inoculated intracranially with Fukuoka-1 strain, and which died at around 120 days post-inoculation, were assayed by this gel-fractionation method and eluted PrP molecules in each fraction were detected by western blot analysis. Oligomeric PrP molecules were well separated from monomers, as predicted. A conventional protease-digestion assay was also performed to detect PrP(res) and revealed that the ratio of PrP(res) to total PrP increased drastically from 105 days. However, the increase of PrP oligomers became significant from 90 days. These PrP oligomers in the early disease stage would, therefore, be sPrP(Sc) molecules that might affect the disease pathology, such as spongiform change and abnormal PrP deposition. We also observed that the resistance of PrP oligomers to proteinase K and insolubility in phosphotungstic acid precipitation increased with disease progression, which suggests that PrP oligomers are not clearly distinguished from cellular PrP or PrP(res) but may overlap in a continuous spectrum. Our study casts light on the ambiguity of the definition of PrP(Sc) and indicates that the abnormality of PrP molecules should be determined from various perspectives, more than protease resistance.