Over-expression of human carcinoma-associated antigen in intrahepatic cholangiocellular carcinoma

Objective: To investigate the expression status of human carcinoma antigen (HCA) in human cholangiocellular carcinomas, and to determine the relationship between HCA and clinical features. Methods: Tissues from 60 intrahepatic cholangiocellular carcinoma (ICC) patients, and normal liver tissues from 20 hepatic hemangioma patients selected randomly were assayed for the expression of HCA by immunohistochemistry, and Western blots. Areas of poorly differentiated (n = 20), moderately-well differentiated (n = 30), highly differentiated tumors (n = 10) from different cases were evaluated. Results were recorded as positive (?5% of cells staining and staining intensity 2+ or 3+) or negative (<5% of cells staining and staining intensity <2+) and analyzed using the χ² test. Results: BCE075 and BDD048 antibodies showed similar staining patterns. The positive immunostaining of BCE075 was mainly localized in the cytoplasm and cell secretions. The staining was positive in 15% of poorly differentiated ICC, 72% of moderately-well differentiated, 100% of highly differentiated tumors. But, staining was not detected in adjacent normal tissue. The differences in HCA expression among these tissues were statistically significant. Also, we found expression of HCA to be closely associated with the degree of differentiation of ICC and tumor cell morphology. There was a correlation between expression of HCA and serum CA19-9. Conclusion: The data suggest that HCA is a potential marker for the diagnosis of cholangiocellular carcinoma.     

High yield expression of non-phosphorylated protein tyrosine kinases in insect cells

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.     

Improved induction of antibodies against key neutralizing epitopes by human immunodeficiency virus type 1 gp120 DNA prime-protein boost vaccination compared to gp120 protein-only vaccination

A major challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development is to elicit potent and broadly neutralizing antibodies that are effective against primary viral isolates. Previously, we showed that DNA prime-protein boost vaccination using HIV-1 gp120 antigens was more effective in eliciting neutralizing antibodies against primary HIV-1 isolates than was a recombinant gp120 protein-only vaccination approach. In the current study, we analyzed the difference in antibody specificities in rabbit sera elicited by these two immunization regimens using peptide enzyme-linked immunosorbent assay and a competitive virus capture assay. Our results indicate that a DNA prime-protein boost regimen is more effective than a protein-alone vaccination approach in inducing antibodies that target two key neutralizing domains: the V3 loop and the CD4 binding site. In particular, positive antibodies targeting several peptides that overlap with the known CD4 binding area were detected only in DNA-primed sera. Different profiles of antibody specificities provide insight into the mechanisms behind the elicitation of better neutralizing antibodies with the DNA prime-protein boost approach, and our results support the use of this approach to further optimize Env formulations for HIV vaccine development.     

Modeling the spontaneous initiation of the polymerization of methyl methacrylate

The mechanism of the spontaneous initiation of the polymerization of methyl methacrylate (MMA) was investigated theoretically. The six minimum energy paths (MEP) of the possible reactions were calculated using the density functional theory (DFT) in conjunction with the B3LYP functional and 6-31G* basis set. The Diels-Alder initiation mechanism (path (I) and path (II)) with remarkably high energy barriers is not applicable to MMA. Four favorable paths were found (path (III), path (IV), path (V) and path (VI)), which are supporting the Flory mechanism. Path (V) has the lowest active energy. Therefore this path is considered as the main path for the spontaneous polymerization of MMA. Figure The mechanism of the spontaneous initiation of the polymerization of methyl methacrylate (MMA) was investigated theoretically. The six minimum energy paths (MEP) of the possible reactions were calculated using the density functional theory (DFT) in conjunction with the B3LYP functional and 6-31G* basis set.     

Diet composition of post-metamorphic bullfrogs (Rana catesbeiana) in the Zhoushan archipelago, Zhejiang Province, China

Bullfrogs (Rana catesbeiana) are listed as one of the 100 worst invasive alien species in the world. They are generalist predators and thus may affect native species through predation. In previous studies, the food contents of bullfrogs were mostly examined at a single site. In the present study, the diet composition of post-metamorphic bullfrogs on eight islands (Daishan, Liuheng, Xiushan, Fodu, Taohua, Xiashi, Cezi, and Putuoshan) in the Zhoushan Archipelago, Zhejiang Province of China, were examined by using the stomach flushing method from June 30 to August 11 in 2005. A total of 391 individual frogs were measured, including 113 adults and 278 juveniles. The analysis of the stomach contents shows that, for adult bullfrogs, the most important prey (by diet volume) overall were Decapoda, Coleoptera, Odonata, Mesogastropoda, Raniformes, and Cypriniformes. For juvenile bullfrogs, these were Decapoda, Coleoptera, Cypriniformes, Odonata, Orthoptera, Hymenoptera, Lepidoptera larvae, Mesogastropoda, and Raniformes. Moreover, the prey size and diet volume increased with the body size of both adult and juvenile bullfrogs. The diet composition of primary preys of bullfrogs was significantly different among the islands. The results indicate that bullfrogs exert different predatory influences on native fauna at different sites and that bullfrogs are generalist predators with extensive ecological impacts on native fauna. __________ Translated from Biodiversity Science, 2006, 14 (5): 363–371 [译自: 生 物多样性]     

Effects of lanthanum on rumen fermentation, urinary excretion of purine derivatives and digestibility in steers

The objective of this study was to evaluate the effects of LaCl₃ supplementation on rumen fermentation, urinary excretion of purine derivatives and feed digestibility in the total tract of steers. Eight ruminally cannulated Simmental steers (420 ± 20 kg) were used in a replicated 4 × 4 Latin square experiment. The treatments were control (without LaCl₃); La-low; La-medium and La-high with 450, 900 and 1800 mg LaCl₃ per steer per day, respectively. Diet consisted of 600 g/kg corn stover and 400 g/kg concentrate (dry matter [DM] basis). Dry matter intake (averaged 9 kg/day) was restricted to a maximum of 90% of ad libitum intake. Ruminal pH (range of 6.59–6.42) was quadratically (P<0.04) changed, whereas total volatile fatty acids (VFA) concentration (range of 74.16–88.61 mM) was linearly (P<0.01) and quadratically (P<0.01) increased with increasing La supplementation. Ratio of acetate to propionate decreased linearly (P<0.01) from 3.28 to 1.79 as La supplementation increased due to the increased in propionate production. In situ ruminal neutral detergent fibre (aNDF) degradation of corn stover was improved but the crude protein (CP) degradability of soybean meal was decreased with increasing La supplementation. Urinary excretion of purine derivatives was quadratically (P<0.01) changed with altering La supplementation (75.5, 81.0, 82.4 and 70.6 mmol/day for control, low-, medium- and high-LaCl₃ supplementation, respectively). Similarly, digestibilities of organic matter, aNDF and CP in the total tract were also linearly and quadratically increased with increasing La supplementation. The present results indicate that supplementation of diet with LaCl₃ improved rumen fermentation and feed digestion in beef cattle. It was suggested that the La stimulated the digestive microorganisms or enzymes in a dose-dependent manner. In the experimental conditions of this trial, the optimum La dose was about 900 mg LaCl₃ per steer per day.     

An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10

The membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 bears the epitopes of two broadly neutralizing antibodies (Abs), 2F5 and 4E10, making it a target for vaccine design. A third Ab, Fab Z13, had previously been mapped to an epitope that overlaps those of 2F5 and 4E10 but only weakly neutralizes a limited set of primary isolates. Here, libraries of Fab Z13 variants displayed on phage were engineered and affinity selected against an MPER peptide and recombinant gp41. A high-affinity variant, designated Z13e1, was isolated and found to be approximately 100-fold improved over the parental Fab not only in binding affinity for the MPER antigens but also in neutralization potency against sensitive HIV-1. Alanine scanning of MPER residues 664 to 680 revealed that N671 and D674 are crucial for peptide recognition as well as for the neutralization of HIV-1 by Z13e1. Ab competition studies and truncation of MPER peptides indicate that Z13e1 binds with high affinity to an epitope between and overlapping with those of 2F5 and 4E10, with the minimal peptide epitope WASLWNWFDITN. Still, Z13e1 remained about an order of magnitude less potent than 4E10 against several isolates of pseudotyped HIV-1. The sum of our molecular analyses with Z13e1 suggests that the segment on the MPER of gp41 between the 2F5 and 4E10 epitopes is exposed on the functional envelope trimer but that access to the specific Z13e1 epitope within this segment is limited. Thus, the ability of MPER-bearing immunogens to elicit potent HIV-1-neutralizing Abs may depend in part on recapitulating the particular constraints that the functional envelope trimer imposes on the segment of the MPER to which Z13e1 binds.