Quantum dots are conventionally applicable for wide-profiling of wall polymer distribution and destruction in diverse cells of rice.

Plant cell walls represent enormous biomass resources for biofuels, and it thus becomes important to establish a sensitive and wide-applicable approach to visualize wall polymer distribution and destruction during plant growth and biomass process. Despite quantum dots (QDs) have been applied to label biological specimens, little is reported about its application in plant cell walls. Here, semiconductor QDs (CdSe/ZnS) were employed to label the secondary antibody directed to the epitopes of pectin or xylan, and sorted out the optimal conditions for visualizing two polysaccharides distribution in cell walls of rice stem. Meanwhile, the established QDs approach could simultaneously highlight wall polysaccharides and lignin co-localization in different cell types. Notably, this work demonstrated that the QDs labeling was sensitive to profile distinctive wall polymer destruction between alkali and acid pretreatments with stem tissues of rice. Hence, this study has provided a powerful tool to characterize wall polymer functions in plant growth and development in vivo, as well as their distinct roles during biomass process in vitro.

Cell-penetrating Peptide-mediated therapeutic molecule delivery into the central nervous system.

The blood-brain barrier (BBB), a dynamic and complex barrier formed by endothelial cells, can impede the entry of unwanted substances - pathogens and therapeutic molecules alike - into the central nervous system (CNS) from the blood circulation. Taking into account the fact that CNS-related diseases are the largest and fastest growing unmet medical concern, many potential protein- and nucleic acid-based medicines have been developed for therapeutic purposes. However, due to their poor ability to cross the BBB and the plasma membrane, the above-mentioned bio-macromolecules have limited use in treating neurological diseases. Finding effective, safe, and convenient ways to deliver therapeutic molecules into the CNS is thus urgently required. In recent decades, much effort has been expended in the development of drug delivery technologies, of which cell-penetrating peptides (CPPs) have the most promising potential. The present review covers the latest advances in CPP delivery technology, and provides an update on their use in CNS-targeted drug delivery.

A high-affinity anti-salbutamol monoclonal antibody: key to a robust lateral-flow immunochromatographic assay.

Among the components that make up a lateral-flow immunochromatographic assay (ICA), antibody is the key. In this paper, salbutamol (SAL) as a model analyte was meticulously designed to prepare immunogen and coating antigen in distinctly different ways. Four hybridoma cell lines were prepared and identified. Among them, C9 had highest affinity, best dose-response behavior, lowest limit of detection, and highest specificity and was chosen to be labeled with colloidal gold as the detector reagent and applied on the conjugate pad. Goat anti-mouse antibody and SAL-BSA conjugate were sprayed on a nitrocellulose membrane as test line and control line, respectively. Under the optimized conditions, the ICA strip was constructed based on a binding inhibition format. Color intensity on the test line was visually distinguishable from that of the negative sample within 5 min, with the visual detection limit of 1 ngml(-1) in phosphate-buffered saline. Cross-reactions with other ß-agonists were not found (<1%). The results from ICA were in a good agreement with those obtained by enzyme-linked immunosorbent assay. The developed ICA has potential as a useful on-site screening tool for SAL in swine urine.

One-step monoclonal antibody based enzyme-linked immunosorbent assay for direct determination of cortisol in serum.

Monoclonal antibodies to cortisol have obvious potential advantages as starting materials for assay systems to detect their levels in body fluids. This is very important for monitoring pituitary gland and adrenal functions. To develop a one-step competitive heterogeneous enzyme-linked immunosorbent assay (ELISA), a monoclonal anti-cortisol antibody was generated using a reasonably designed haptenic derivative. Cortisol-3-O-carboxymethyloxime was coupled to carrier protein bovine serum albumin (BSA) to enhance its immunogenicity. Spleen cells were prepared from a BALB/c mouse, which had repeatedly been immunized with a conjugate of cortisol-3-O-carboxymethyloxime-bovine serum albumin (cortisol-3-O-CMO-BSA), to be fused with SP2/0 myeloma cells. After one fusion experiment, four hybridoma clones secreting a practical antibody were established. One of the resulting monoclonal antibodies, 2C9D11B5, showed an affinity constant (Ka) of 1.4 × 10(10) M(-1) for cortisol and provided a practical calibration curve (limit of detection [LOD], 0.26 ng per assay) in this ELISA system employing cortisol-21-hemisuccinate-horseradish peroxidase (cortisol-21-HS-HRP) as a tracer. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (3.5%), cortisone (0.47%), corticosterone (<0.01%), progesterone (<0.01%), 17-hydroxyprogesterone (1.2%), 6-hydroxycortisol (7.6%), and tetrahydrocortisol (<0.01%). The intra-assay and inter-assay coefficient of variations (CVs) ranged from 4.3% to 9.2% and 3.8% to 10.4 %, respectively. The analytical recoveries were 92.3% to 116.3%. Serum cortisol levels of healthy volunteers were determined after chilled acetone, stripped to be 292.76 ± 201.38 ng/mL (n=5), which are in the reference range.

Development of a direct competitive enzyme-linked immunosorbent assay using a sensitive monoclonal antibody for bisphenol A.

To set up an immunoassay-based method to detect Bisphenol A (BPA), we generated a monoclonal antibody (MAb) using a specially designed carboxyl derivative of BPA as the immunogen. BPA-HS was synthesized by reaction using BPA and succinic anhydride. The mice were immunized with the BPA-HS-BSA conjugate. The MAb was obtained from a hybridoma. In addition, we showed that the MAb was highly specific for BPA. The limit of detection was approximately 0.05 ng mL(-1) (ppb) in assay buffer and 0.1 ng mL(-1) (ppb) in water samples. The recoveries of BPA for water samples were from 90.8% to 114%, and coefficients of variation were from 15.6% to 39.4%. Thus, the ELISA method is a rapid and high throughput screening tool to detect BPA in water products.

Development of a sensitive monoclonal antibody-based ELISA for the detection of sulfamethazine in cow milk, honey, and swine urine.

A specific monoclonal antibody (MAb) against sulfamethazine was produced with hybridoma technology. This assay shows very high sensitivity with IC50 of 0.4 ng/mL and LOD of 0.05 ng/mL when it was run in 0.02 mol/L PBS (pH 7.5). This MAb has shown high specificity to sulfamethazine, with little cross reactivity for sulfamerazine (5.27%) and sulfadimethoxypyrimidine (1.12%) and very low cross reactivity values for the other test compounds (≤0.1%). Sulfamethazine was spiked in milk, honey, and swine urine. After a simple extraction procedure the extracts at appropriate dilution were analyzed by ELISA. Satisfactory results were obtained by this assay, with average recoveries of 95-116% and coefficients of variation (CVs) of 5-9%. These results suggests that the MAb-based ELISA will be a feasible quantitative/screening method for sulfamethazine residue in real samples.

SOCS-1 protects against Chlamydia pneumoniae-induced lethal inflammation but hampers effective bacterial clearance.

Suppressor of cytokine signaling 1 (SOCS1) plays a major role in the inhibition of STAT1-mediated responses. STAT1-dependent responses are critical for resistance against infection with Chlamydia pneumoniae. We studied the regulation of expression of SOCS1 and SOCS3, and the role of SOCS1 during infection with C. pneumoniae in mice. Bone marrow-derived macrophages (BMM) and dendritic cells in vitro or lungs in vivo all showed enhanced STAT1-dependent SOCS1 mRNA accumulation after infection with C. pneumoniae. Infection-increased SOCS1 mRNA levels were dependent on IFN-alphabeta but not on IFN-gamma. T or B cells were not required for SOCS1 mRNA accumulation in vivo. Infection-induced STAT1-phosphorylation occurred more rapidly in SOCS1(-/-) BMM. In agreement, expression of IFN-gamma responsive genes, but not IL-1beta, IL-6, or TNF-alpha were relatively increased in C. pneumoniae-infected SOCS1(-/-) BMM. Surprisingly, C. pneumoniae infection-induced IFN-alpha, IFN-beta, and IFN-gamma expression in BMM were attenuated by SOCS1. C. pneumoniae infection of RAG1(-/-)/SOCS1(-/-) mice induced a rapid lethal inflammation, accompanied by diminished pulmonary bacterial load and increased levels of iNOS and IDO but not IL-1beta, IL-6, or TNF-alpha mRNA. In summary, C. pneumoniae infection induces a STAT1, IFN-alphabeta-dependent and IFN-gamma independent SOCS1 mRNA accumulation. Presence of SOCS1 controls the infection-induced lethal inflammatory disease but impairs the bacterial control.

Protective effects of green tea polyphenols in the 6-OHDA rat model of Parkinson's disease through inhibition of ROS-NO pathway.

BACKGROUND: Nitric oxide (NO) and related pathways are thought to play an important role in the pathogenesis of Parkinson's disease (PD). Our in vitro experiments suggested that green tea polyphenols (GTP) might protect dopamine neurons through inhibition of NO and reactive oxygen species (ROS). METHODS: Immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling assay, electron spin resonance spin trapping, enzyme linked immunosorbent assay, and molecular biological methods were used to investigate the effects of GTP in an unilateral 6-hydroxydopamine (6-OHDA)-treated rat model of PD. RESULTS: GTP treatment dose-dependently protected dopaminergic neurons by preventing from midbrain and striatal 6-OHDA-induced increase in 1) both ROS and NO levels, 2) lipid peroxidation, 3) nitrite/nitrate content, 4) inducible nitric oxide synthase, and 5) protein-bound 3-nitro-tyrosine. Moreover, GTP treatment dose-dependently preserved the free radical scavenging capability of both the midbrain and the striatum. CONCLUSIONS: These results support the in vivo protection of GTP against 6-OHDA and suggest that GTP treatment might represent a neuroprotective treatment of PD.

Role of IRAK4 and IRF3 in the control of intracellular infection with Chlamydia pneumoniae.

TLR signal transduction involves a MyD88-mediated pathway, which leads to recruitment of the IL-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Toll/IL-1R translation initiation region domain-containing adaptor-inducing IFN-beta-mediated pathway, resulting in the activation of IFN regulatory factor (IRF)3. Both pathways can lead to expression of IFN-beta. TLR-dependent and -independent signals converge in the TNF receptor-associated factor 6 (TRAF6) adaptor, which mediates the activation of NF-kappaBeta. Infection of murine bone marrow-derived macrophages (BMM) with Chlamydia pneumoniae induces IFN-alpha/beta- and NF-kappaBeta-dependent expression of IFN-gamma, which in turn, will control bacterial growth. The role of IRAK4 and IRF3 in the regulation of IFN-alpha/beta expression and NF-kappaBeta activation was studied in C. pneumoniae-infected BMM. We found that levels of IFN-alpha, IFN-beta, and IFN-gamma mRNA were reduced in infected IRAK4(-/-) BMM compared with wild-type (WT) controls. BMM also showed an IRAK4-dependent growth control of C. pneumoniae. No increased IRF3 activation was detected in C. pneumoniae-infected BMM. Similar numbers of intracellular bacteria, IFN-alpha, and IFN-gamma mRNA titers were observed in C. pneumoniae-infected IRF3(-/-) BMM. On the contrary, IFN-beta(-/-) BMM showed lower IFN-alpha and IFN-gamma mRNA levels and higher bacterial titers compared with WT controls. C. pneumoniae infection-induced activation of NF-kappaBeta and expression of proinflammatory cytokines were shown to be TRAF6-dependent but did not require IRAK4 or IRF3. Thus, our data indicate that IRAK4, but not IRF3, controls C. pneumoniae-induced IFN-alpha and IFN-gamma secretion and bacterial growth. IRAK4 and IRF3 are redundant for infection-induced NF-kappaB activation, which is regulated by TRAF6.

Protein nitration increased by simulated weightlessness and decreased by melatonin and quercetin in PC12 cells.

A variety of experiments suggest that space flight is associated with an increase in oxidative stress in organism. To explore the effects of oxidative stress on neuronal cells during microgravity, we used rat pheochromocytoma (PC12) cells as a neuronal cell model, cultured in a clinostat, which could simulate microgravity, to investigate the effects of reactive nitrogen species on protein nitration in PC12 cells during clinorotation. The effects of melatonin and quercetin on protein nitration in PC12 cells were also assayed to evaluate the possible protective role of melatonin or quercetin as an antioxidant. The results of immunological staining showed that after the 3 days' clinorotation the protein expressions of neuronal nitric oxide synthase and inducible nitric oxide synthesis were up-regulated. Our data also reflected that the concentrations of nitric oxide and nitrotyrosine were significantly increased after clinorotation, and they were reduced markedly in cells that were treated with 50 micromol/L melatonin or 0.5 micromol/L quercetin during simulated microgravity, when compared to those of control cells. These results suggest that clinorotation-induced weightlessness increases oxidative stress responses in PC12 cells, and melatonin or quercetin was shown to protect PC12 cells from oxidative damage during simulated weightlessness.

Preparation and identification of anti-2, 4-dinitrophenyl monoclonal antibodies.

2, 4-Dinitrophenyl (DNP) is a widely used hapten in molecular biology and immunoassay fields. Considering that 2, 4-dintrophenylhydrazine (DNPH) could be used as DNA probe and bind with protein carbonyl to form a stable 2 4-dinitrophenyl (DNP) hydrazone product, on which the level of oxidative stress could be validated with a sensitive noncompetitive ELISA, we prepared DNP-aminocaproic acid and NHS-aminocaproic acid-dinitrobenzene and the conjugates between DNP and carrier proteins such as bovine thyroglobulin (BTG) and bovine serum albumin (BSA). High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, twenty stable murine monoclonal antibodies (MAbs) producing cell lines to DNP were generated. The donor mouse produced antiserum with a high titer of 1/1,280,000. Five MAbs were selected for further characterization as class and subclass. After four successive limiting dilutions, antibodies were produced by five clones with high affinities ranging from 10(10) to 10(11) M(-1). These clones were found to be of IgG(1) subclass with kappa and lambda light chain. Competitive ELISA and SPR-based sensing system for the detection of DNPH are both used to confirm the specificity of MAb (4D(9)A(9)C(2)C(2)).

Preparation and identification of monoclonal antibodies against daintain.

Daintain is a 17-kDa polypeptide originally purified from porcine intestine. This polypeptide is associated with insulin secretion and inflammatory responses. Daintain is highly similar in amino acid sequence to allograft inflammatory factor-1 (AIF-1). Here we report the preparation and identification of monoclonal antibodies (MAbs) against daintain. To enhance its immunogenicity, daintain was coupled to carrier protein bovine serum albumin (BSA) by a two-step glutaraldehyde method. Using conventional procedures, we obtained four stable hybridoma cell lines that can produce and secret anti-daintain MAbs. We further analyzed their isotypes, titer, and affinity, and found that those MAbs belong to the G1 subclass with kappa light chains. The MAbs were capable of recognizing daintain as determined by Western blotting. The produced MAbs will be a useful tool for further investigation of daintain functions in organisms.

Preparation and identification of anti-melatonin monoclonal antibodies.

Anti-melatonin monoclonal antibodies (MAbs) of high titer were prepared by coupling melatonin to bovine serum albumin with formaldehyde and by immunizing BALB/c mice with multifocal intradermal injections and by fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Five MAbs were selected for further characterization as classes and subclasses. After four successive limiting dilutions, antibodies were produced by these five clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin Ig G1 and IgG(2b) subclass with kappa light chain. A systematic study of cross-reactions with seven compounds (indole, aromatic and imidazole derivatives) showed that the antibody had a high specificity for melatonin, low reactivity with 6-hydroxymelatonin and N-acetyl-5-hydroxytryptamine, and no detectable reactivity with tryptamine, l-tryptophan, 5-methoxytryptamine and N-acetyl-L-tryptophan. The roles of the indole nucleus and the side chain in the determination of the antigenic properties of the molecule are discussed. One of the MAbs, 4C9D7, was used to establish a competitive enzyme-linked immunosorbent assay for the detection of melatonin in supernatant.

Development and identification of monoclonal antibodies against parathion.

An amino-derivative of parathion was prepared and conjugated to human serum albumin (HSA) and bovine thyroglobulin (BTG) via diazonium condensation. Spleen cells producing high titer antibody were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, we generated nine stable murine monoclonal antibodies (MAbs) producing cell lines to parathion. After four successive limiting dilutions, antibodies produced by nine clones had high affinities, ranging from 10(9) to 10(12) M(-1). These clones were found to be of IgG class and IgM class with k light chain. Subclass determination showed that the clones produced IgG(1), IgG(2a), IgG(2b), and IgM types of antibody. One clone (2H(9)) was used to establish the calibration curve with a sensitivity of 26 ng/mL, a practical working range of 46.8-6000 ng/mL parathion.

Preparation and identification of monoclonal antibodies against pea albumin 1b (PA 1b).

PA 1b (pea albumin 1b), extracted from pea seeds, is thermostable and is multifunctional. It has an attractive peros toxicity, and is also involved in the regulation of callus growth and cell proliferation. Here we report the preparation of monoclonal antibodies (MAbs) against this peptide for further investigation of peptide distribution and functions. PA 1b was coupled to carrier protein using the two-step glutaraldehyde method as an immunal antigen. Five stable cell lines producing anti-PA 1b MAbs were obtained. We analyzed their isotypes, titer, and affinity and found that those MAbs belong to the G(1) and G(2b) subclasses with kappa light chain, respectively. Using these antibodies, a competitive inhibition ELISA was developed, and approximately 15 nmol/L of antigen was detected.

[Preparation of polyclonal and monoclonal antibodies against CRF and change of CRF in the sleep-deprived rat brain].

AIM: To produce polyclonal and monoclonal antibodies against corticotropin-releasing factor (CRF) and to study the changes of CRF in sleep-deprived rat brain with the antibodies acquired. METHODS: Commercial CRF was linked to bovine throglobulin (BTG) and bovine serum albumin (BSA) respectively to produce immunogen and embedding antigen. New Zealand rabbits and BALB/c mice were immunized with the BTG-CRF immunogen to produce polyclonal and monoclonal antibodies, respectively. The acquired antibodies were appraised with ELISA and immnohistochemical staining. The characterized antibodies were used to observe the changes of CRF in the rat brain 48 h after sleep deprivation. RESULTS: CRF polyclonal antibody and 9 clones of monoclonal antibodies were obtained with high titer, affinity and specificity. Among them, the polyclonal antibody and 2 monoclonal antibodies (1D10, 2F4) were excellent in immunocytochemical staining. The CRF-like immunoreactive substances were found more strongly expressed in the neurons of paraverticular hypothalamic nucleus (PVN), central subnucleus of amygdala (CeA), oval subnucleus of bed nucleus of stria terminalis (BNSTov), and nucleus of solitary tract (NTS) in sleep-deprived rat brain. While they were much weaker and even absent in the control. CONCLUSION: The polyclonal and monoclonal antibodies against CRF were successfully produced for immunocytochemical studies. The results indicate that CRF may play an important role in stress-responsive modulation during sleep deprivation. PVN, CeA and BNSTov are integral part of brain circuit related to the stress modulation.

[Detection of small molecules based on surface plasmon (correction of plasman) resonance technology].

OBJECTIVE: To develop a detective method applied in online assaying of astronauts' humours using the portable online bio-molecules analyzer (POBA) based on surface plasmon resonance biosensor. METHOD: An assay format was developed based on the detection of 2, 4-Dinitrophenyl-hydrazine. The bio-molecule slide was made by DNP-BSA. Range of detection and standard curve were obtained using inhibition assay. Reliability and specificity of the assay were also tested. RESULT: 1) The linear range of the assay was 7.8 ng/ml-2 micrograms/ml with lower detection limit of 2.5 ng/ml; 2) Preparation of the bio-molecule slide and regeneration of the biosensor ensured detections for many samples. CONCLUSION: This assay method can be used to detect small molecules sensitively, rapidly and easily. It can be repeated with good reliability, and has a good application in space medicine.

Group-selective immunoassay for the detection of morphine in urine.

A new competitive inhibition immunoassay (group-selective immunoassay; GSI) has been developed to detect free morphine in urine with the Fab' fragments of monoclonal antibodies (MAbs) (1B(12)F(9)B(4), IgG(1), kappa, K(aff) = 9.66 x 10(10)M(-1)). At the first assay step, microtiter plates were coated with morphine-ovalbumin (M-6-S-OVA), in which free amino acids were protected by a glutaraldehyde cross-linking modification. The modification did not essentially influence the antibody-binding capacity of the immunosorbent. At the second assay step, anti-morphine MAbs' Fab' fragments, in which free amino groups were biotinylated by N-hydrosuccinimide-biotin ester, were bound to chemically modified immunosorbent. The biotin residues were then detected by the streptavidin-peroxide conjugate. This method has a sensitivity of 3.50 x 10(-15) mol/L using very little volume of sample, covering up to almost 1.20 x 10(-11) mol/L of standard concentration of morphine with good reproducibility. Standard curve prepared in urine indicated a good correlation between the concentration of morphine and the value of OD (y = 1/ax + b; r = 0.99939257, S = 0.01138127). Coefficients of variation for this immunoassay were 1.41 approximately 6.61% within-a-day assay and 2.31 approximately 8.99% between days assay. The recoveries were 94 approximately 101.4% from negative urine and 95.2 approximately 107.5% from positive urine samples, respectively. This method has application as a specific screen for morphine in drug abusers, to study the metabolism of the drug in the body, or to screen the monoclonal antibodies (MAbs) against morphine.

A novel competitive ELISA for both free and protein-bound nitrotyrosine.

3-Nitro-L-tyrosine (nitrotyrosine) has recently been considered to be useful as a biomarker of endogenous production of several reactive nitrogen species including peroxynitrite. In the present study, nitrotyrosine was coupled to human serum albumin (HSA) using a two-step glutaraldehyde method and immunized mouse with multifocal intradermal injections. Using a conventional immunization protocol, 12 stable monoclonal antibodies (MAbs) producing cell lines recognizing nitrotyrosine were obtained. Six MAbs were selected for further characterization. A study of cross-reactions with nitrotyrosine-like compounds showed that the antibodies had a high specificity for nitrotyrosine, but no detectable reactivity with L-tyrosine, p-nitro-L-phenylalanine, o-phospho-L-tyrosine or 3-amino-L-tyrosine. Using these high titer and affinity antibodies, a competitive inhibition ELISA was developed with a lower detection limit of approximately 20 nmol/L to detect both free and protein-bound nitrotyrosine in biological systems.

[Changes of endocrine hormones during -6 degrees head down bed rest in human].

OBJECTIVE: To investigate changes of endocrine hormones during 7 d head down bed rest (HDBR). METHOD: Six healthy male volunteers served as the subjects and experienced 7 d -6 degrees HDBR. Urine was collected from 6:00-22:00 and from 22:00-6:00. Serum was collected 48 h before HDBR, 48 h and 120 h during HDBR. Then the endocrine indices in urine and serum were determined. RESULT: 1) The levels of serum CORT and ALD increased at 48 h during HDBR, while serum T3, T4, TP, UN decreased but they all recovered to normal at 120 h during HDBR. 2) The level of urine CORT, ALD and NE reached its peak in 24-48 h, and then gradually decreased to normal level. CONCLUSION: The endocrine indices in serum and urine changed in the early period but returned to normal level gradually with the proceeding of HDBR.