Preparation of artificial antigen and egg yolk-derived immunoglobulin (IgY) of florfenicol amine for ELISA assay.

To investigate the feasibility of IgY technology for the immune detection of haptens, a specific egg yolk antibody (IgY) has been developed in order to detect the florfenicol amine (FFA) residues. FFA was conjugated to bovine serum albumin (BSA) by glutaraldehyde (GA) and the conjugate was used to immunize laying chickens. Anti-FFA IgY antibody was purified by PEG-6000 precipitation and identified by SDS-PAGE. The titer of anti-FFA IgY antibody reached a peak of 1:128,000 after three booster injections. Checkerboard titration showed that a 1:800 dilution of anti-FFA IgY could give an optical density (OD) at around 1.0 at 10 µg/mL FFA-OVA coating concentration. An indirect competitive enzyme-linked immunosorbent assay (icELISA) using specific anti-FFA IgY showed that the IC50 value of anti-FFA IgY was 12.30 ng/mL and the regression curve equation was y = -13.71x + 64.95 (R (2) = 0.945). The strategy of developing anti-hapten IgY antibody is that it may be further used as a new reagent for an immunoassay of hapten residues.

MS2 Virus-like Particles as a Versatile Peptide Presentation Platform: Insights into the Deterministic Abilities for Accommodating Heterologous Peptide Lengths.

Virus-like particles (VLPs) are nanostructures with the potential to present heterologous peptides at high density, thereby triggering heightened immunogenicity. RNA bacteriophage MS2 VLPs are a compelling delivery platform among them. However, a notable hurdle arises from the immune response toward MS2 coat protein, swiftly eliminating subsequent vaccinations via the same vector. Although larger inserts effectively mask carrier epitopes, current research predominantly focuses on displaying short conserved peptides (<30 aa). A systematic evaluation regarding the deterministic ability of MS2 VLPs as a platform for presenting heterologous peptides remains a gap. In light of this, we employed the "single-chain dimer" paradigm to scrutinize the tolerance of MS2 VLPs for peptide/protein insertions. The results unveiled functional MS2 VLP assembly solely for inserts smaller than 91 aa. Particularly noteworthy is the largest insertion achieved on the MS2 VLPs to date: the RNA helicase A (RHA) dsRNA-binding domains (dsRBD1). Attempts to introduce additional linkers or empty coat subunits fail to augment the expression level or assembly of the MS2 VLPs displaying dsRBD1, affirming 91 aa as the upper threshold for exogenous protein presentation. By illuminating the precise confines of MS2 VLPs in accommodating distinct peptide lengths, our study informs the selection of appropriate peptide and protein dimensions. This revelation not only underscores the scope of MS2 VLPs but also establishes a pivotal reference point, facilitating the strategic manipulation of MS2 VLPs to design next-generation epitope/antibody-based therapeutics.

Molecular cloning and characterization of the gene encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4.

The gene bgaP encoding cold-active beta-galactosidase from a psychrotrophic and halotolerant Planococcus sp. L4 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the BgaP gene revealed an open reading frame of 2031 bp encoding for a protein of 677 amino acid residues. The BgaP was heterologously expressed in E. coli and purified followed by Ni2+ affinity chromatography. The molecular mass of the native enzyme was approximately 156 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the BgaP indicated molecular masses of 78 and 77.311 kDa, respectively, suggesting that the BgaP is a dimer. The purified BgaP had an isoelectric point of 4.8 and exhibited maximal activity at 20 degrees C and pH 6.8 under the assay conditions used. The enzyme is particularly thermolabile, losing all activity in only 10 min at 45 degrees C. It was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG); the Km values with ONPG and lactose were calculated to be 5.4 and 20.4 mM at 5 degrees C, respectively. The catalytic efficiencies of BagP for lactose at 5 and 20 degrees C had 14 and 47 times more than that of E. coli beta-galactosidase at 20 degrees C, respectively. Therefore, cold-active beta-galactosidase from the psychrotrophic and halotolerant Planococcus sp. L4 could conceivably be developed to fulfill the practical requirements to enable its use for lactose removal in milk and dairy products at low temperature or a reporter enzyme for psychrophilic genetic systems.

Biodegradation of benzene and its derivatives by a psychrotolerant and moderately haloalkaliphilic Planococcus sp. strain ZD22.

The potential for biodegradation of aromatic hydrocarbons simultaneously at low temperatures and under saline and alkaline conditions is not well understood, but such biodegradation would be useful for remediation of polluted sites. A psychrotolerant, moderately haloalkaliphilic pure culture using benzene as a sole source of carbon and energy was isolated by selective enrichment from alkaline and saline soils in the vicinity of the Daqing oil field in China. An analysis of the 16S rDNA gene sequence and morphological and physiological characteristics showed that this strain is a member of the genus Planococcus, and it was designated as strain ZD22. Strain ZD22 could grow at temperatures between 2 and 36 degrees C (pH 7.5-11) and salt concentrations from 0.5 to 25%. Its optimal conditions for biodegradation of benzene were 20 degrees C (pH 9.5) and 10% salt concentration. Strain ZD22 not only utilized benzene, toluene, ethylbenzene and o-xylene, but also degraded chlorobenzene, bromobenzene, iodobenzene and fluorobenzene. The kinetic model of strain ZD22 for benzene was solved to obtain mumax=0.34 h-1, Ks=0.041 mM, n=1.21, Sm=10.2 mM. To our knowledge, this is the first report of biodegradation of benzene and its derivatives simultaneously under multiple extreme conditions.

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112.

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.

Changes of interleukin expression correlate with Helicobacter pylori infection and lymph node metastases in gastric carcinoma.

Helicobacter pylori (HP) infection induces expression of IL-8 and IL-10 in benign gastric epithelium. This study compared the expression of cytokines in CD4+ and CD8+ lymphocyte subsets of peripheral blood lymphocytes (PBL), benign mucosal lymphocytes (ML), and tumor infiltrative lymphocytes (TIL) as well as in the benign and malignant epithelial cells of the same patient, with respect to the presence of HP infection, lymph node metastases, and tumor histologic type. The mRNA of the cytokines was measured by a semiquantitative RT-PCR method. The levels were ranked and compared using the Wilcoxon sign-ranked test. Compared with CD8+ ML, the CD8+ TIL expresses higher levels of IL-6 and IL-8 but lower level of IL-4 in patients with lymph node metastases. In patients with HP infection, expression of IL-8 and IL-10 was higher in the gastric carcinoma cells than in the benign epithelial cells while expression of IL-6 and IL-8 were higher in CD8+ TIL than CD8+ ML. Overexpression of IL-8 in HP associated gastric carcinomas suggested that they might have arisen from HP-infected epithelial cells.

Biological properties of bone marrow-derived early and late endothelial progenitor cells in different culture media.

Ex vivo expansion of endothelial progenitor cells (EPCs) may be a promising strategy to overcome the clinical problem of limited cell numbers. As the culture medium is the key for the cell characteristics, the effects of different culture media on EPCs were investigated in the present study. Rat bone marrow mononuclear cells were cultured in different media, including M-199 media with 20% fetal bovine serum (FBS) and bovine pituitary extract (M1); M-199 media with 10% FBS, 20 ng/ml vascular endothelial growth factor (VEGF) and 10 ng/ml basic fibroblast growth factor (bFGF; M2) or epidermal growth medium (EGM)-2MV media. The cell morphology and biological functions, such as proliferation, adhesion, migration, tube formation and nitric oxide (NO) production were subsequently assayed in vitro. Moreover, endothelial biomarkers and apoptosis were also analyzed. The results showed that endothelial­like cells appeared in all of the culture systems. First­passage cells, namely early EPCs, tended to form colonies in M2 and EGM-2MV media but showed a fusiform shape in M1 media. The 3rd or 4th generation EPCs, namely late EPCs, cultured in EGM-2MV media exhibited increased adhesion, migration, tube formation and NO production as compared with EPCs in M1 or M2 media. Furthermore, late EPCs cultured in EGM-2MV expressed higher levels of endothelial cell markers, such as von Willibrand factor (vWF)and CD31, but relatively greater levels of apoptosis were observed. In conclusion, cell culture conditions, for example the medium used, affects the biological properties of bone marrow-derived early and late EPCs.

Study on the anticancer activity of coumarin derivatives by molecular modeling.

Protein kinase 2 (CK2) is a potential target, and the coumarins were identified as the attractive CK2 inhibitors. In this study, two models (CoMFA and CoMSIA) were established, and their reliabilities were supported by statistical parameters. From the CoMFA and CoMSIA models, the hydrophobic and hydrogen bonds play very important roles in the interactions between inhibitors and CK2, which were confirmed sufficiently by molecular docking. Furthermore, the binding mode of the inhibitors at the active sites of CK2 was also investigated by docking study. The hydroxyl at the position R(5) is more important for coumarins inhibitors because it forms hydrogen bonds not only with Lys68 as hydrogen acceptor but also with H(2) O as hydrogen donor. In addition, hydroxyl can make electrostatic interactions with electropositive Lys68 residue. The large group at the R(6) position is not conducive to inhibitor dock into the groove of the binding site of CK2. When there is nitro group, the electrostatic interaction between ligand and receptor is enhanced significantly, and the nitro oxygen can form hydrogen bonds with the backbone NH of Lys68 and Asp175 simultaneously. The results obtained from molecular modeling techniques not only provide the models to predict the activity of inhibitors but also lead to a better understanding of the interactions between inhibitors and CK2, which will be very helpful for drug design.