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Mussel foot proteins (Mfps), which help mussels attach to various surfaces, are considered to be promising biomaterials due to their outstanding adhesive properties. However, limited production and lack of post-translational modifications of tyrosine residues into 3,4-dihydroxyphenylalanine (Dopa) in bacterial expression systems have hampered their applications. In the present study, for the first time we established the expression of recombinant Mytilus galloprovincialis foot protein type 3 variant B (fp-3B) in Escherichia coli; and achieved its viable production (~51 mg/L). Additionally, the Dopa content and adhesive properties of fp-3B co-expressed using various types of tyrosinases were compared. Consequently, the co-expression of fp-3B construct together with tyrosinase from Verrucomicrobium spinosum (TyrVs) yielded up to 87 mg/L of modified fp-3B; hydroxylation of tyrosine residues accounted for 57.18% by acid-borate difference spectroscopy. The modified fp-3B also showed significant coating and adhesive ability, and its bulk-scale adhesive strength was 2.9-fold higher than that of unmodified fp-3B. Compared with other type 3 mussel foot proteins, the high-yield expression and extensive hydroxylation level of the recombinant protein indicate that fp-3B co-expressed with TyrVs (3B-Vs) has the potential to be widely used as bioglues.

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (Ki) of 1.69 × 10-8 mol·L-1. Further study showed that HMEI-A inhibited the formation of neutrophil extracellular trap (NET). These results suggested that HMEI-A from the leech of H. manillensis is a novel elastase inhibitor which can suppress NET formation. It may play a significant role in blood-sucking of leeches and is a potential candidate as an anti-inflammatory agent.

Background In children, focal segmental glomerulosclerosis (FSGS) is the main cause of steroid resistant nephrotic syndrome (SRNS). To identify specific candidates and the mechanism of steroid resistance, we examined the formalin-fixed paraffin embedded (FFPE) renal tissue protein profiles via liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods Renal biopsies from seven steroid-sensitive (SS) and eleven steroid-resistant (SR) children FSGS patients were obtained. We examined the formalin-fixed paraffin embedded (FFPE) renal tissue protein profiles via liquid chromatography tandem mass spectrometry (LC-MS/MS). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology (GO) analysis, as well as the construction of protein-protein interaction (PPI) network were performed. Two proteins were further valiadated by immunohistochemistry staining in FSGS patients and mice models. Results In total, we quantified more than 4000 proteins, of which 325 were found to be differentially expressed proteins (DEPs) between the SS and SR group (foldchange ≥2, P Conclusions In this study, the potential mechanism and candidates related to steroid resistance in children FSGS patients were identified. It could be helpful in identifying potential therapeutic targets and predicting outcomes with these proteomic changes for children FSGS patients.

The content and speciation of heavy metals can fundamentally affect the hydrolysis of sludge. This research study investigates the migration and transformation rule of heavy metals during the hydrolysis process by measuring the content of exchangeables (F1), bound to carbonates (F2), bound to Fe-Mn oxides (F3), bound to organic matter (F4), and residuals (F5) under different periods of time undergoing hydrolysis. The results show that the hydrolysis process generally stabilized Cu, Zn, Mn, Ni, Pb, Cr, and As by transforming the unstable states into structurally stable states. Such transformations and stabilization were primarily caused by the changes in local metal ion environment and bonding structure, oxidation of sulfides, pyrolyzation of organic matter, and evaporation of resulting volatile materials. An X-ray diffractometry (XRD) of the residuals conducted after hydrolysis indicated that hydrolysis did have a significant influence on the transportation and transformation of heavy metals.

Ultraviolet (UV) irradiation is a common form of DNA damage that can cause pyrimidine dimers between DNA, which can cause gene mutations, even double-strand breaks and threaten genome stability. If DNA repair systems default their roles at this stage, the organism can be damaged and result in disease, especially cancer. To better understand the cellular response to this form of damage, we applied highly sensitive mass spectrometry to perform comparative proteomics of phosphorylation in HeLa cells. A total of 4367 phosphorylation sites in 2100 proteins were identified, many of which had not been reported previously. Comprehensive bioinformatics analysis revealed that these proteins were involved in many important biological processes, including signaling, localization and cell cycle regulation. The nuclear pore complex, which is very important for RNA transport, was changed significantly at phosphorylation level, indicating its important role in response to UV-induced cellular stress. Protein-protein interaction network analysis and DNA repair pathways crosstalk were also examined in this study. Proteins involved in base excision repair, nucleotide repair and mismatch repair changed their phosphorylation pattern in response to UV treatment, indicating the complexity of cellular events and the coordination of these pathways. These systematic analyses provided new clues of protein phosphorylation in response to specific DNA damage, which is very important for further investigation. And give macroscopic view on an overall phosphorylation situation under UV radiation.

Biological control is a promising measure in the control of plant disease. In the present study, we isolated 13 endophytic strains from Angelica dahurica. Among them, an endophytic strain which was named GYLH001 exhibited remarkable activity against Magnaporthe oryzae. 16S rRNA sequence analysis, biochemical and physiological proved that it is Bacillus tequilensis. The sterilized culture filtrate of GYLH001 can inhibit the growth of M.oryzae, which suggests the presence of secondary metabolites. Proved by experiment, GYLH001 can produce cellulase, protease, gelatinase, indole-3-acetic acid and 1-amino-cyclopropane-1-carboxylate deaminase. In addition, the temperature experiment showed that secondary metabolites produced by GYLH001 had good thermal stability. They can remain activity even heated at 100°C for 30 min. They also had good acid-resistance in heavily acidic condition. But under alkaline condition, the antifungal effect decreased significantly. By simulative field tests, the spraying of GYLH001 spore solution could prevent and treat rice blast. Through continuous separation and purification of sterilized culture filtrate and identification by mass spectrometry, the molecular weight of an active substance is 364.26. In the control of rice blast, B. tequilensis GYLH001 has potential as a biological control agent in agriculture.

Ethnopharmacological relevance Da-Bu-Yin-Wan (DBYW) and Qian-Zheng-San (QZS), two traditional Chinese herbal formulas, were clinically employed to treat Parkinson's disease (PD) for decades. Aim of the study Our previous studies demonstrated neuroprotective effects of DBYW and QZS on mitochondrial function in mice model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In present research, we aimed to investigate the possible neuroprotective mechanisms of DBYW and QZS. Materials and methods The effects of DBYW and QZS on the behavioral changes (pole test), expression of tyrosine hydroxylase (TH) of substantia nigra by immunohistochemistry, monoaminergic contents and activity of striatum by high performance liquid chromatography, neuronal ultrastructure changes by transmission electron microscopy, mitochondrial DNA (mtDNA) damage by long-extension polymerase chain reaction (PCR), and mRNA expression of mitochondrial subunit NADH dehydrogenase 1(ND1) by qualitative real-time PCR were investigated. Results Present study demonstrated that DBYW and QZS not only ameliorated the behavior induced by the administration of MPTP and synergistically prevented the decreasing of TH expression, but also increased monoaminergic contents and activity, improved the ultrastructural changes, decreased the mtDNA damage, and synergistically up-regulated the expression of ND1 in mRNA level. Conclusions These results suggest that DBYW and QZS possess anti-parkinsonism and neuroprotective properties.

The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2) treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998), Lon proteases (dr0349 and dr1974), NADH-quinone oxidoreductase (dr1506), thiosulfate sulfurtransferase (dr2531), the DNA repair protein UvsE (dr1819), PprA (dra0346), and RecN (dr1447), are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.

Excessive accumulation of iron in the body can lead to organ injury. Some flavonoids which possess high affinity to iron can be used as iron chelators for curing iron overload diseases such as haemochromatosis. In this article, the effects of baicalin and quercetin on different mouse tissues (liver, kidney and heart) afflicted with iron overload-induced injury were studied. It was found that after administration of 500 mg kg⁻¹ iron dextran for 45 days, the degree of oxidative injury in the three organs was different. When iron dextran-induced iron-overloaded mice were fed a diet containing baicalin or quercetin (1% w/w), both flavonoids showed significant effects in suppressing iron overload-induced injury. The efficiencies of both flavonoids on different tissues varied. Both flavonoids caused a significant decrease of iron content in liver and kidney tissues but not in the heart, whereas the flavonoids were more efficient in inhibiting the increase of carbonyl content and the thiobarbituric acid reactive substance levels in heart tissues. The results indicate that the protective effects of baicalin and quercetin on different tissues of iron-overloaded mice are different.

A new functional nanoparticle, consisting of a silica core onto which short-chain zwitterions are chemically connected, was successfully prepared and showed excellent antifouling performance to protein solutions. These nanoparticles (NPs) own excellent stability even in 1M NaCl solutions for at least 48 h. The interaction between these "zwitterated" NPs and proteins were investigated by dynamic light scattering (DLS), turbidimetric titration, and isothermal titration calorimetry (ITC). The results demonstrated that the zwitterated NPs had antifouling property both in single protein solutions and serum (fetal bovine serum, FBS). The zwitterated NPs also own abundant functional groups which could conjugate with biomolecules for future applications in therapeutic and diagnostic field.

Both cinnamomin and ricin are type II ribosome-inactivating proteins. Cinnamomin is less cytotoxic compared with ricin. In order to clarify the mechanism of their different cytotoxicities, the interaction of cinnamomin and its A-chain with model membrane was investigated and compared with that of ricin and its A-chain. It was revealed that cinnamomin is less effective than ricin in interacting with model membrane. Cinnamomin A-chain interacts with model membrane much less violently than ricin A-chain. The differences in the interaction of cinnamomin, ricin or their A-chains with model membrane might at least in part indicate the different cytotoxicity between cinnamomin and ricin.

Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can specifically act on the universally conserved sarcin/ricin domain (S/R domain) of the largest RNA in ribosome and thus irreversibly inactivate ribosome for protein synthesis. Cinnamomin is a multifunctional type II RIP isolated in our laboratory from the mature seeds of the camphor tree. This protein has been extensively studied with regard to its purification, characteristics, structure and function, genetic expression, enzymatic mechanism, physiological role in seed cell and toxicity to cancer cells and insect larvae. The research results of cinnamomin obtained in our laboratory are summarized in this review. Understanding of cinnamomin and the relative new proteins will help expand our knowledge of RIPs and may accelerate theoretical study and the development of their potential applications.

Cinnamomin, a type 11 ribosome-inactivating protein (RIP), was isolated from the mature seeds of camphor tree (Cinnamomum camphora). In this paper, small amount of free A- and B-chain of cinnamomin were found to be present in the mature seed cell of C. camphora besides the intact cinnamomin. Our results demonstrated that camphorin, a type I RIP previously reported to coexist with cinnamomin in the seeds of C camphora, actually was the A-chain of cinnamomin. The percentage of free A- and B-chain in the total cinnamomin was 2.6-2.8% in the seed extract. Of these free A- and B-chain approximate 80% already existed in the seed cell, only about 20% were produced during the purification operation. As the enzymatic activity to reduce disulfide bond of cinnamomin in the seed extract of C camphora was detected, we proposed that the free A- and B-chain were derived from the enzymatic reduction of the interchain disulfide bond of cinnamomin. It was demonstrated that the endogenous type 11 RIPs of several plant species, such as Cinnamomum porrectum, Cinnamonium bodinieri and Ricinus communis, could be enzymatically reduced into the free A- and B-chain in their respective seed cells. The function of the free A-chain in the seed cell and the possibility that metabolic enzymes might be involved in the reduction of the interchain disulfide bond of type 11 RIPs in vivo are discussed. (C) 2003 Elsevier Science Ltd. All rights reserved.

Poly(e-L-lysine) (e-PL) and poly(L-diaminopropionic acid) (PDAP) co-production by Streptomyces albulus PD-1 from cane molasses and hydrolysate of strepyomyces cells (HSC) was investigated for the first time in this study. The optimal initial total sugar concentration of the cane molasses pretreated with sulfuric acid was determined to be 20 g L(-1), and HSC could substitute for yeast extract for e-PL and PDAP co-production. When fed-batch fermentation was performed in 1t fermentor with pretreated cane molasses and HSC, 20.6 ± 0.5 g L(-1) of e-PL and 5.2 ± 0.6 g L(-1) of PDAP were obtained. The amount of strepyomyces cells obtained in one fed-batch fermentation is sufficient to prepare the HSC to satisfy the demand of subsequent fermentations, thus the self-cycling of organic nitrogen source becomes available. These results suggest that the low-cost cane molasses and HSC can be used for the economical production of e-PL and PDAP by S. albulus PD-1.

Polymeric brushes have emerged as a novel 3D material platform that provides great amounts of binding sites for biomolecules. This paper investigates the covalent immobilization mechanism of protein by spherical poly(acrylic acid) brushes (SPAABs) in the widely adopted N-hydroxysuccinimide/N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide hydrochloride (NHS/EDC) process. It was discovered that electrostatic interaction plays a crucial role in the covalent immobilization of protein. Due to the existence of 3D architecture and "Donnan effect", SPAABs exhibit quite different immobilization kinetics in comparison with conventional 2D materials. Under conditions favorable to electrostatic interaction, the effect of "electrostatic interaction induced covalent binding" was observed as a result of competitive immobilization by physical adsorption and chemical binding. On the basis of the mechanism study, a new "chemical conjugation after electrostatic entrapment" (CCEE) method was developed which set the chemical and physical immobilization process apart. A more effective and well-defined covalent immobilization was achieved. And the binding capacity can be tuned in a wide range (0-4.2 mg protein/mg SPAABs) with a high level of control.

High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven genes (Glu-1Aix1 ∼ 4 and Glu-1Aiy1 ∼ 3) encoding the seven HMW-GSs were isolated using PCR technique with degenerate primers, and confirmed by bacterial expression and Western blotting. Sequence analysis indicated that the seven Ag. intermedium HMW-GSs shared high similarity in primary structure to those of wheat, but four of the seven subunits were unusually small compared to the representatives of HMW-GS from wheat and two of them possessed extra cysteine residues. The alignment and clustering analysis of deduced amino acid sequences revealed that 1Aix1 and 1Aiy1 subunits had special molecular structure, belonging to the hybrid type compounding between typical x- and y-type subunit. The xy-type subunit 1Aix1 is composed of the N-terminal of x-type and C-terminal of y-type, whereas yx-type subunit 1Aiy1 comprises the N-terminal of y-type and C-terminal of x-type. This result strongly supported the hypothesis of unequal crossover mechanism that might generate the novel coding sequence for the hybrid type of HMW-GSs. In addition to the aforementioned, the other novel characteristics of the seven subunits were also discussed. Finally, phylogenetic analysis based on HMW-GS genes was carried out and provided new insights into the evolutionary biology of Ag. intermedium.

We studied the interaction between proteins and polystyrene-polyacrylic acid (PS-PAA) spherical polyelectrolyte brushes with different polyacrylic acid (PAA) chain lengths, including the physical adsorption and chemical adsorption in PBS buffer. Results showed that the amount of bovine serum albumin (BSA) physically adsorbed on PS-PAA spherical polyelectrolyte brushes decreased to a minimum of 33 microg/mg whereas the amount of streptavidin (SA) chemically adsorbed increased with the increase of chain length and carboxyl quantity. The biotin binding capacity of streptavidin chemically adsorbed on PS-PAA spherical polyelectrolyte brushes was roughly evaluated via enzyme competitive inhibition.

The excellent optical properties of quantum dots (QDs), such as high brightness, high photostability, continuous absorption, and narrow emission bandwidth, make them ideal as optical labels to develop QD-based immunohistofluorescence (IHF) imaging for multiplexing cancer biomarker detection on formalin-fixed and paraffin-embedded (FFPE) tissues. IHF is very important for the prediction of a patient's response to cancer chemotherapy or radiotherapy. QD-based IHF faces several challenges that differ from those encountered by organic dye based IHF for clinical assays. The current work addresses some of these issues. Initially, the chemical stability of QDs and organic dyes were compared. The results showed that QDs were stable for at least 5 months on FFPE tissue, whereas organic dyes were photobleached shortly after exposure to light. Various staining methods were also studied. QD fluorescence intensity on the tissue stained with primary antibody (Ab, p16, survivin, EF1α) conjugated QDs from our company was comparable to the signal from a commercially available method in which the tissue was stained with a primary p16 Ab and a QD-labeled secondary goat anti mouse Ab respectively. Finally, the effect of the amount of Ab conjugated to QD on tissue imaging was also studied. There was no significant increase in the QD fluorescence signal on tissues when the Ab:QD ratio increased from 5 to 30. In addition, protein G was tested as an adaptor protein to link Ab to QDs for IHF staining. However, the proper blocking of the protein G on QDs was necessary to reduce crosstalk. The biomarker quantification in QD-based IHF was validated by conventional Western blot and immunohistochemistry. The results contained herein demonstrate a promising application of QDs in multiplex detection and quantification of biomarkers.

A novel pH-responsive coating technique was developed and applied to CE successfully in this paper. The coating was formed by bonding mixed opposite charge poly(acrylic acid) and poly(2-vinylpyridine) randomly onto the inner wall of a silica capillary. The coating processes were first characterized by ellipsometry and atomic force microscopy at macroscale and microscale, respectively. Measurements of EOF were implemented to confirm the coating. Direction and velocity of EOF became controllable from negative to positive, showing a perfect sigmoidal curve as the coating net charges alternated by the pH of BGE. The control of the EOF makes it possible to analyze different kinds of small molecules, peptides, and proteins successfully in the same capillary. Results showed that the stability and reproducibility for separations of fluoroquinolone standards were satisfactory for more than a hundred separations. A series of basic and acidic protein standards were separated with admirable efficiency and minimal adsorption using both polarities. The separation of tryptic BSA digest showed that the prepared capillary has immense potential in analyzing a single sample with both acidic and basic separations, which achieved the expectation in proteomics study by CE-MS.

Mast cells play a central role in asthma. Moreover, serum miRNA-221-3p (miR-221) has been shown to be markedly increased in children with asthma. In the current study, we aimed to examine miR-221 expression in an asthma model and elucidate the mechanisms regulating interleukin (IL)-4 secretion in mast cells. Using polymerase chain reaction, we found that miR-221 was upregulated in a murine asthma model and in P815 mast cells after lipopolysaccharide (LPS) stimulation. Moreover, miR-221 upregulated IL-4 secretion from P815 cells, as shown by enzyme-linked immunosorbent assays. Bioinformatics analysis, luciferase reporter gene assays, and western blotting showed that phosphatase and tensin homolog (PTEN) was a target of miR-221 and could block IL-4 secretion stimulated by miR-221. The phosphorylation of p38 (protein) and activity of nuclear factor-kappaB (NF-κB) were increased after overexpression of miR-221, as shown by electrophoretic mobility shift assays. Finally, treatment with specific inhibitors could block IL-4 secretion. In conclusion, miR-221, which was overexpressed in a murine asthma model, stimulated IL-4 secretion in mast cells through a pathway involving PTEN, p38, and NF-κB.

To overcome major challenges of non-specific protein adsorption on nanoparticles for nanosensing and nanodiagnosis, an efficient method for robust chemical modification was developed to achieve excellent specific biorecognition and long-term stability in complex biomedia. This method is demonstrated by a highly specific and sensitive immunoassay (IA), using superparamagnetic nanospheres (NSs) with high magnetite content. The non-specific protein adsorption on the NSs was suppressed dramatically when modified with dual functional poly(carboxybetaine methacrylate) (polyCBMA) via surface-initiated atom transfer radical polymerization (SI-ATRP) and chemically grafted with antibodies of the β subunit of human chorionic gonadotrop (anti-β-hCG). The response to hCG of IA NSs with polyCBMA coatings was highly consistent in either phosphate-buffered saline (PBS) or 50% fetal bovine serum (FBS), which is far less variable than the response of the IA NSs without polyCBMA coatings. After all, a very robust platform for IA NSs with excellent specific biorecognition was obtained. It is expected that this method for nanoparticle modification could be widely used in ultrasensitive nanosensing and nanodiagnosis in the future.

Affinity chromatography has played an increasingly important role both in the pharmaceutical industry and academic research. In the present study, we report our preliminary investigation on the relationship between the affinity ligand structure and its adsorption to multi-protein samples. The structure of the ligands, including the size of the ring (cyclic group) and the length of the chain (linear group), has a great impact on the adsorption of ligands to proteins. Meanwhile, the functional groups that the ligands carry are also closely related to the adsorption of ligands to proteins. This research provides good guidance for the design and synthesis of affinity materials in affinity chromatography. It is also useful to other protein-ligand interaction-related research.

Background: Environmental stress can accelerate the directional selection and evolutionary rate of specific stress-response proteins to bring about new or altered functions, enhancing an organism's fitness to challenging environments. Plateau pika (Ochotona curzoniae), an endemic and keystone species on Qinghai-Tibetan Plateau, is a high hypoxia and low temperature tolerant mammal with high resting metabolic rate and non-shivering thermogenesis to cope in this harsh plateau environment. Leptin is a key hormone related to how these animals regulate energy homeostasis. Previous molecular evolutionary analysis helped to generate the hypothesis that adaptive evolution of plateau pika leptin may be driven by cold stress. Methodology/Principal Findings: To test the hypothesis, recombinant pika leptin was first purified. The thermogenic characteristics of C57BL/6J mice injected with pika leptin under warm (23+/-1 degrees C) and cold (5+/-1 degrees C) acclimation is investigated. Expression levels of genes regulating adaptive thermogenesis in brown adipose tissue and the hypothalamus are compared between pika leptin and human leptin treatment, suggesting that pika leptin has adaptively and functionally evolved. Our results show that pika leptin regulates energy homeostasis via reduced food intake and increased energy expenditure under both warm and cold conditions. Compared with human leptin, pika leptin demonstrates a superior induced capacity for adaptive thermogenesis, which is reflected in a more enhanced beta-oxidation, mitochondrial biogenesis and heat production. Moreover, leptin treatment combined with cold stimulation has a significant synergistic effect on adaptive thermogenesis, more so than is observed with a single cold exposure or single leptin treatment. Conclusions/Significance: These findings support the hypothesis that cold stress has driven the functional evolution of plateau pika leptin as an ecological adaptation to the Qinghai-Tibetan Plateau.

To purify recombinated GNLY which is expressed by prokaryotic expression system, and to prepare the monoclonal antibody (mAb) against it.The solvable protein was purified by affinity chromatography. And employing the fusion protein GNLY immuned BALB/c mice, using conventional hybridoma technology prepared the mAb against human GNLY. Then purified and determined for titer by indirect ELISA method, for antigenic epitopes by additive ELISA, for relative affinity index by sulfocyanate elution method, and analyzed for subclass, specificity and stability.We got the soluble reactive fusion GNLY, and its purity and content were 95%, 0.8 g/L, respectively. Four cell strains secreting mAb against human GNLY were screened, 6C8, 9C6, 5G7and5E5. Their neutralizing titers were 1:100-1:3 200 and (0.1 - 8) x 10(-4) in supernatant and ascites.We successfully purified the fusion protein of GNLY, and prepared the mAb against human GNLY, lying a certain foundation for its laboratory and clinical research.

The increased distribution of microcystins (MCs) has become a global issue. However, the MC response machinery has not been completely studied and elucidated until now. Our previous dose-related analysis has revealed that different concentrations of mirocystin-RR (MC-RR) can trigger comprehensive and various alterations in the protein expression profile using a proteomic approach. To further dissect the kinetics of cellular responses associated with MC-RR cytotoxic effects, the present study has set out to investigate the temporal protein expression pattern at three time points (3, 12, and 24 h after exposure). The relative intensity has changed significantly in MC-RR-treated FL cells, as compared with the control, in a total of 127 protein spots. One hundred and two proteins have been further identified with high confidence by peptide mass fingerprinting. These proteins can be categorized into diverse functional classes such as signal transduction, apoptosis, protein degradation, cell cycle, cell differentiation, transporter, and so forth. The wide range of different proteins involved suggests that MC-RR has profound effects on the biological response and toxic consequences of the affected cells. The identification of p53 and its potential targets confirms a known role for p53 in the MC-RR response. Moreover, it is noteworthy that MC-RR can up-regulate the expression of the PP2A A subunit and down-regulate the expression of a number of proteins implicated in the ubiquitin-proteasome pathway. Therefore, the present expression data provide a global view of dynamic changes in cell responses to MC-RR and, more importantly, generate novel hypotheses regarding MC-RR-responsive mechanisms.

Microcystins (MC), the potent inhibitor of protein phosphatase 1 and 2A, are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promoting activity have not been fully elucidated. To better understand the mechanisms underlying microcystin-RR (MC-RR) induced toxicity as well as provide the possibility for the establishment of biomarkers for MC-RR exposure, differential proteome analysis on human amnion FL cells treated by MC-RR was carried out using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Image analysis of silver-stained 2-dimensional gels revealed that 89 proteins showed significant differential expression in MC-RR treated cells compared with control, and 8 proteins were unique to MC-RR treated cells and 8 proteins were only detected in control cells. Sixty-six proteins were further identified with high confidence by peptide mass fingerprinting. Some of the identified differentially expressed proteins have clearly relationship with the process of apoptosis, signal transduction, and cytoskeleton alteration which are consistent with the literature. The functional implications of alterations in the levels of these proteins were discussed. However, most of which have not been reported previously to be involved in cellular processes responded to MC-RR. Therefore, this work will provide new insight into the mechanism of MC-RR toxicity.

Cinnamomin is a type II ribosome-inactivating protein (RIP) and its A-chain (CTA) is a RNA N-glycosidase. It is observed that modification of tyrosine residues by N-acetylimidazole (N-AI) causes almost complete loss of CTA activity. Adenine partially protects tyrosine residues from modification by N-AI. It is proposed that tyrosine residues are involved in the active site of CTA and they are crucial in recognition and binding of ribosomal RNA. Tryptophan residues of CTA are also studied by NBS modification.

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.

Differentiation of neuronal precursor cells in response to neurotrophic differentiation factors is accompanied by the activation of membrane-anchored SNT signaling adaptor proteins. Two classes of differentiation factors, the neurotrophins and fibroblast growth factors, induce rapid tyrosine phosphorylation of SNT1(FRS2alpha), which in turn enables SNT1 to recruit Shp2 tyrosine phosphatase and Grb2 adaptor protein in complex with the Ras GDP/GTP exchange factor Sos. To determine effector functions of SNT that promote neuronal differentiation of PC12 pheochromocytoma cells, we engineered a chimeric protein, SNT1(IRS)CX, bearing the effector region of SNT1 and the insulin receptor recognition domains of IRS2. Insulin promoted tyrosine phosphorylation of SNT1(IRS)CX in transfected PC12 cells accompanied by sustained activation of ERK1/2 mitogen-activated protein kinases and neuronal differentiation. The SNT1(IRS)CX-mediated response was dependent on endogenous Ras, MEK, and Shp2 activities. Mutagenesis of SNT1(IRS)CX identified three classes of effector motifs within SNT critical for both sustained ERK activation and neuronal differentiation: 1) four phosphotyrosine motifs that mediate recruitment of Grb2, 2) two phosphotyrosine motifs that mediate recruitment of Shp2, and 3) a C-terminal motif that functions by helping to recruit Sos. We discuss possible mechanisms by which three functionally distinct SNT effector motifs collaborate to promote a downstream biochemical and biological response.