Establishment of a chicken monoclonal antibody panel against mammalian prion protein

A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A total of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scrapie-infected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cleavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP antibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.     

Cellular functions of mitogen-activated protein kinases and protein tyrosine phosphatases in ovarian granulosa cells

A number of endocrine and paracrine factors regulate the follicular growth and atresia, which are closely associated with granulosa cell survival and apoptosis. However, the molecular mechanisms underlying the intracellular events induced by these factors are poorly understood. Here, we describe the correlation of mitogen-activated protein kinase (MAPK) activities with granulosa cell survival and apoptosis, and the cellular functions of protein tyrosine phosphatases (PTPs) in these cells based on our recent data. MAPKs play key roles in various cellular responses because numerous extracellular stimuli are integrated into MAPKs. The protein phospho-Tyr level regulated by protein tyrosine kinases (PTKs) and PTPs is a major control mechanism for processes as diverse as cell survival, proliferation, differentiation, and metabolism. Although PTKs are critically involved in granulosa cell survival and proliferation, there are no reports indicating the roles of PTPs in the ovary except for ours. Information about MAPKs and PTPs in these cells will provide a basis for the understanding of the molecular mechanisms controlling the fate of follicles.     

Isolation and characterization of hematopoietic progenitor cells in canine bone marrow

For ultimate diagnoses of canine leukemia or malignant lymphoma, we sought to isolate hematopoietic progenitor cells (HPCs) from canine bone marrow (BM) using physiological phenotypes. Canine BM cells were separated by equilibrium discontinued density centrifugation, and HPCs, detected by in vitro colony formation, were significantly enriched in the relatively low density (LD) fraction. In flow cytometry, many CD34 or MHC class II expressing cells were detected in the LD fraction, but these were not significantly enriched. When the LD cells were separated, using a cell-sorting method, into cells with high affinity of wheat germ agglutinin (WGAhigh) and cells with WGAlow, almost all multipotent HPCs (MHPCs) and HPCs committed to myeloid lineage were found in the WGAhigh population. When the WGAhigh population was further stained for rhodamin 123, almost all MHPCs were included in the dull population (Rhlow), but not in the bright one (Rhhigh). Morphologically, most Rhlow cells were round, blastic cells containing a large nucleus with nucleoli and narrow cytoplasm. Based on these results, we suggest that all of the MHPCs in canine BM show the Rhlow WGAhigh LD phenotype, and may contain hematopoietic stem cells, which are the primitive HPCs.     

Antioxidant activity of dietary fruits, vegetables, and commercial frozen fruit pulps

Fruits, vegetables, and commercial frozen pulps (FP) consumed in the Brazilian diet were analyzed for antioxidant activities using two different methods, one that determines the inhibition of copper-induced peroxidation of liposome and another based on the inhibition of the co-oxidation of linoleic acid and beta-carotene. The anthocyanin-rich samples showed the highest, concentration-dependent, antioxidant activities in both systems. In the liposome system, at both 10 and 50 microM gallic acid equivalent (GAE) addition levels, the neutral and acidic flavonoids of red cabbage, red lettuce, black bean, mulberry, Gala apple peel, jambolao, acai FP, mulberry FP, and the acidic flavonoids of acerola FP showed the highest antioxidant activities (>85% inhibition). In the beta-carotene bleaching system, the samples cited above plus red guava gave inhibition values >70%. On the other hand, some samples showed pro-oxidant activity in the liposome system coincident with a low antioxidant activity in the beta-carotene system. There was no relationship between total phenolics content, vitamin C, and antioxidant activity, suggesting that the antioxidant activity is a result of a combination of different compounds having synergic and antagonistic effects.     

Artoindonesianin L, a new prenylated flavone with cytotoxic activity from Artocarpus rotunda

A new prenylated flavone, named artoindonesianin L (1), was isolated from Artocarpus rotunda (Hout) Panzer (Moraceae). Its structure was elucidated as on the basis of spectroscopic evidence. Along with this new compound, four known phenolic compounds were also isolated from this plant and identified as artonins M (2) and E (3), cycloartobiloxanthone (4) and artonin O (5). All these compounds showed significant cytotoxicity against murine P388 leukemia cells.     

Photosynthesis and photoassimilate transport during root hypoxia in Melaleuca cajuputi, a flood-tolerant species, and in Eucalyptus camaldulensis, a moderately flood-tolerant species

We compared the photosynthetic and photoassimilate transport responses of Melaleuca cajuputi Powell seedlings to root hypoxia with those of Eucalyptus camaldulensis Dehnh. Control and hypoxia treated roots were maintained in a nutrient solution through which air or nitrogen was bubbled. Under root hypoxic conditions, seedlings of M. cajuputi, a flood-tolerant species, maintained height growth, whereas seedlings of E. camaldulensis, a moderately flood-tolerant species, showed markedly decreased height growth compared with control seedlings. Root hypoxia caused decreases in whole-plant biomass, photosynthetic rate and stomatal conductance in E. camaldulensis, but not in M. cajuputi. Photoassimilate transport to roots decreased significantly in E. camaldulensis seedlings 4 days after treatment and starch accumulated in mature leaves. Photoassimilate supply to hypoxic roots of E. camaldulensis seedlings was, thus, limited by reduced photoassimilate transport rather than by reduced photosynthesis. In contrast, M. cajuputi seedlings showed sustained photoassimilate transport to hypoxic roots and persistent photosynthesis, which together provided a substantial photoassimilate supply to the roots. Sucrose accumulated in hypoxic E. camaldulensis roots, but not in hypoxic M. cajuputi roots. A stable, low sucrose concentration in hypoxic roots would let M. cajuputi seedlings prolong photoassimilate transport to the roots. Photoassimilate partitioning among the water-soluble carbohydrates, starch and structural carbohydrates within the roots was unaffected by root hypoxia in E. camaldulensis, but in M. cajuputi, partitioning was shifted somewhat from structural carbohydrates to water-soluble carbohydrates. This suggests that M. cajuputi seedlings are able to increase photoassimilate utilization in metabolism and sustain energy production under root hypoxic conditions.