Production and characterization of monoclonal antibodies against human ceruloplasmin

Ceruloplasmin (CP) is the major plasma antioxidant and copper transport protein. Monoclonal antibodies (mAbs) against human CP were produced and characterized. A total of five hybridoma cell lines were established (CP2, CP10, CP20, CP25, CP30). From the epitope mapping analysis, two subgroups of mAbs recognize different peptide fragments were identified. When the purified CP was incubated with the mAbs, the ferroxidase activity of CP was inhibited up to a maximum 57 %. Immunoblotting with various tissue homogenates indicated that all the mAbs specifically recognize a single protein band of 130 kDa. They also appear to be extensively cross-reactive among different mammalian including human and avian sources. These results demonstrated that only one type of immunologically similar CP is present in all of the mammalian tissues including human. The CP mAbs could be of great benefit to design the diagnostic kit for CP-related diseases such as Wilson's disease.     

Quercetin decreases the expression of ErbB2 and ErbB3 proteins in HT-29 human colon cancer cells

Quercetin has chemoprotective properties in experimental colon cancer models, and in vitro studies have demonstrated that quercetin inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 receptor tyrosine kinases have been associated with the development of human colon cancer, and the expressions of both receptors are high in HT-29 cells. In this study, we assessed quercetin regulation of HT-29 and SW480 cell apoptosis and the influence of quercetin on the protein expression of ErbB2, ErbB3, Akt, Bax and Bcl-2. We cultured HT-29 cells in the presence of various concentrations (0, 25, 50, or 100 micromol/L) of quercetin or rutin. Quercetin inhibited HT-29 cell growth in a dose-dependent manner, whereas rutin had no effect on the cell growth. DNA that was isolated from cells treated with 50 micromol/L of quercetin exhibited an oliogonucleosomal laddering pattern characteristic of apoptotic cell death. Western blot analysis of cell lysates revealed that Bcl-2 levels decreased dose-dependently in cells treated with quercetin, but Bax remained unchanged. Quercetin increased levels of cleaved caspase-3 and the 89-kDa fragment of poly (ADP-ribose) polymerase. In addition, phosphorylated Akt levels were markedly lower in cells treated with 25 micromol/L quercetin, but total Akt levels decreased only at 100 micromol/L quercetin. Furthermore, a dose-dependent decrease in ErbB2 and ErbB3 levels was detected in quercetin-treated cells. The results obtained using SW480 cells were similar to those obtained with HT-29 cells. In conclusion, we have shown that quercetin inhibits cell growth and induces apoptosis in colon cancer cells, and that this may be mediated by its ability to down-regulate ErbB2/ErbB3 signaling and the Akt pathway.     

SKI306X, an oriental herbal mixture, suppresses gastric leukotriene B4 synthesis without causing mucosal injury and the diclofenac-induced gastric lesions

SKI306X compound is a herbal mixture. This plant was in oriental medicine and was clinically approved for the treatment of osteoarthritis (OA) in Korea. SKI306X was previously found to have anti-inflammatory, analgesic and cartilage protective effects in several experimental models. In this study, SKI306X was investigated for its gastro-sparing effects on the gastric mucosa comparing with those of diclofenac, a conventional NSAID, and celecoxib, a cyclooxygenase-2 (COX-2) specific inhibitor. To investigate acute gastric damaging properties of SKI306X, the stomach of the animals was histologically and immuno-histochemically examined after single or repeated administration, and SKI306X demonstrated excellent gastric tolerability. SKI306X did not cause significant gastric irritation, erosion, or ulceration up to the orally administered dose of 2 g/kg and the intraperitoneal (i.p.) dose of 125 mg/kg. In contrast, diclofenac caused mucosal erosion, ulceration and bleeding at clinically effective doses. To determine the mode of gastro-sparing action, eicosanoid synthesis was examined in gastric mucosa and blood. SKI306X significantly decreased gastric and blood leukotriene B(4) (LTB(4)) production. However, SKI306X showed either no effect or a slight increase in levels of prostaglandin E(2) (PGE(2)). In addition, gastro-protective effects of SKI306X were exhibited by suppressing diclofenac-induced erosion and ulceration of gastric mucosa in a rat model and the possible mechanism of these effects were investigated. These studies demonstrated that SKI306X did not produce any significant damage up to dose of 2 g/kg and was effective in significantly protecting the damage associated to diclofenac-induced gastric ulcerations. SKI306X could spare the gastric mucosa through significantly suppressing gastric leukotriene (LT) synthesis.     

Descending influences from the lateral hypothalamus and amygdala converge onto medullary taste neurons

The lateral hypothalamus (LH) and the central nucleus of the amygdala (CeA) exert an influence on many aspects of ingestive behavior. These nuclei receive projections from several areas carrying gustatory and viscerosensory information, and send axons to these nuclei as well, including the nucleus of the solitary tract (NST). Gustatory responses of NST neurons are modulated by stimulation of the LH and the CeA, and by several physiological factors related to ingestive behavior. We investigated the effect of both LH and CeA stimulation on the activity of 215 taste-responsive neurons in the hamster NST. More than half of these neurons (113/215) were modulated by electrical stimulation of the LH and/or CeA; of these, 52 cells were influenced by both areas, often bilaterally. The LH influenced more neurons than the CeA (101 versus 64 cells). Contralateral stimulation of these forebrain areas was more often effective (144 responses) than ipsilateral (74). Modulatory effects were mostly excitatory (102 cells); 11 cells were inhibited, mostly by ipsilateral LH stimulation. A subset of these cells (n = 25) was examined for the effects of microinjection of DL-homocysteic acid (DLH), a glutamate receptor agonist, into the LH and/or CeA. The effects of electrical stimulation were completely mimicked by DLH, indicating that cell somata in and around the stimulating sites were responsible for these effects. Other cells (n = 25) were tested for the effects of electrical stimulation of the LH and/or CeA on the responses to taste stimulation of the tongue (32 mM sucrose, NaCl and quinine hydrochloride, and 3.2 mM citric acid). Responses to taste stimuli were enhanced by the excitatory influence of the LH and/or CeA. These data demonstrate that descending influences from the LH and CeA reach many of the same cells in the gustatory NST and can modulate their responses to taste stimulation.     

Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self‐assembly that aligns lysine residues for crosslinking. As a result, both the full‐length monomer as well as elastin‐like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full‐length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self‐assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full‐length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 392–407, 2013.     

Microfabricated particulate drug-delivery systems

Micro- and nanoparticulate drug-delivery systems (DDSs) play a significant role in formulation sciences. Most particulate DDSs are scaffold-free, although some particles are encapsulated inside other biomaterials for controlled release. Despite rapid progress in recent years, challenges still remain in controlling the homogenicity of micro-/nanoparticles, especially for two crucial factors in particulate DDSs: the size and shape of the particles. Recent approaches make use of microfabrication techniques to generate micro-/nanoparticles with highly controllable architectures free of scaffolds. This review presents an overview of a burgeoning field of DDSs, which can potentially overcome some drawbacks of conventional techniques for particle fabrication and offer better control of particulate DDSs.     

Internal structure of the postcapillary high-endothelial venules of rodent lymph nodes and Peyer's patches and the transendothelial lymphocyte passage

Scanning electron microscopy (SEM) shows that the postcapillary high-endothelial venules of lymph nodes and Peyer's patches consist of two segments each with a different surface relief: a proximal segment with a cobblestone surface pattern and a distal segment of interlacing cytoplasmic plates. Both segments have deep adluminal crevices in which lymphocytes are lodged. The internal structural configuration of this endothelium has been examined by transmission electron microscopy (TEM) of serial sections of lymph nodes and Peyer's patches of mice, rats, and guinea pigs. The serial sections revealed that the endothelial cell bodies and their cytoplasmic extensions were disposed in a direction generally lateral to the luminal surface and intruded into the intercellular spaces of similarly disposed neighboring endothelial cells, resulting in a complex interlacing cellular pattern. Lymphocytes penetrated the endothelial cell body and secondarily followed an intracellular pathway through which they entered the extravascular compartment. At the exposed surfaces of the adluminal venule wall, recirculating lymphocytes were seen in SEM images to enter the endothelium by penetrating the endothelial cell body. The mode of migration of lymphocytes lodged in the endothelial crevices could be determined by SEM and has been examined by TEM of serial sections. At these locations as at the exposed surfaces, lymphocytes also entered the venule by penetrating the endothelial cell body. At both sites this transcellular pathway was followed by lymphocyte entry into the intercellular spaces from which they migrated into the extravascular compartment.     

Transformed T0 orchardgrass (Dactylis glomerata L.) plants produced from highly regenerative tissues derived from mature seeds

A highly efficient and reproducible transformation system for orchardgrass (Dactylis glomerata L. cv. Rapido, 2n=42=28) was established using microprojectile bombardment of highly regenerative, green tissues derived from mature seeds. These tissues, induced from embryogenic callus, were bombarded with a mixture of three plasmids containing the hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and #-glucuronidase (uidA; gus) genes. From 147 individual explants bombarded, 11 independent hygromycin-resistant lines (7.5%) were obtained after an 8- to 16-week selection period using 30-50 mg/l hygromycin B. Of the 11 independent lines, ten (91%) were regenerable. The presence and integration of the transgene(s) were assessed using PCR and DNA blot hybridization. Coexpression frequency of the three transgenes (hpt/bar/uidA) in T₀ plants was 20%, and of two transgenes, either hpt/bar or hpt/uidA, 45-60%. Due to greenhouse conditions optimized for the growth of other species, T₁ seed has not been obtained from these plants. While the inability to analyze progeny plants precludes the conclusive demonstration of stable transformation, the results of all molecular and biochemical analyses of T₀ plants are consistent with the production of stably transformed plants. Frequent change in ploidy level was observed in transformed T₀ orchardgrass plants. Plants from only three of the ten independent lines analyzed had the normal tetraploid number of chromosomes (2n=42=28), while plants from seven lines (70%) were octaploid (2n=82=56). The octaploid plants had abnormal morphological features, such as narrower, thicker and more upright leaves.