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.

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.

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 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.

[Space flight and peroxidative damage].

Space flight is associated with an increase of peroxidative damage after returning to 1 g. The effect is more pronounced after long-duration space flight and can even last for several weeks after landing. In humans there is increased lipid peroxidation in erythrocyte membranes, reduced blood antioxidants, and increased urinary excretion of 8-iso-prostaglandin F2alpha, and 8-oxo-7, 8 dihydro-2 deoxyguanosine. Isoprostane 8-iso-prostaglandin F2alpha and 8-oxo-7, 8 dihydro-2 deoxyguanosine are markers for oxidative damage to lipids and DNA, respectively. The changes are attributed to a combination of energy deficiency that occurs during flight and substrate competition for amino acids occurring between repleted muscle and other tissues during the recovery phase. The observations in humans have been complemented by studies in rodents, which showed increased production of lipid peroxidation products and decreased antioxidant enzyme activity afterflight. The changes in rodents were attributed to the stress associated with re-entry into Earth's gravity. Reducing the imbalance between the production of endogenous oxidant defenses and oxidant production by increasing the supply antioxidants in diet may lessen the severity of the postflight increase in oxidative stress.

[Advances in monoclonal antibody researches].

Monoclonal antibody techniques are very important tools in modern life science research. Despite extensive research efforts paid in recent years, and promising results yielded in the study on the structure and function of genes and proteins, there is still a great need for further researches on the definition, principle and applicability of some immunological methods. This review gives an overview of the advances in immunological researches, including DNA immunization, cellular immunization and preparation of monoclonal antibodies. Using methods of modem molecular immunology, such as genetic immunization, cellular immunization, subtractive immunization and repetitive immunization multiple sites (RIMMS), to construct eukaryotic expression vector and to prepare high-affinity monoclonal antibodies in short time, the conventional method which is time-consuming and laborious could be improved. It is meaningful to the field of basic research and application, such as proteomics, biochip, clinical medicine and diagnosis and therapy of diseases.