In this study, we examined the development of the upper eyelids to provide a basic understanding of gross anatomical structures and information relative to mechanisms of congenital anomalies in the upper eyelids. We studied the upper eyelids by external and histological observation in 48 human embryos and in fetuses from 5 to 36 weeks postfertilization. The upper eyelid fold began to develop at Stage 18. Upper and lower eyelids fused from the lateral cantus at Stage 22, and fusion was complete by 9 weeks of development. Mesenchymal condensations forming the orbital part of the orbicularis oculi (OO), tarsal plate, and the eyelashes and their appendages, were first seen at Week 9. Definite muscle structures of the upper eyelid, such as the orbital part of the OO and the levator palpebrae superioris and its aponeurosis, and the Mü ller's muscle were observed at 12 and 14 weeks, respectively. In addition, orbital septum, arterial arcade and orbital fat pad, and tarsal gland (TG) were apparent at 12, 14, and 18 weeks, respectively. Opening of the palpebral fissure was observed at Week 20. In addition, we defined the directional orientation between the levator aponeurosis and orbital septum and the growth pattern of the TG. Our results will be helpful in understanding the normal development of the upper eyelid and the origins of upper eyelid birth defects. Anat Rec, 294:789-796, 2011. V V C 2011 Wiley-Liss, Inc.

Background: Wnt signalling dysregulation has been implicated in cancer, including colon and gastric cancer. Initiation of Wnt signalling is modulated by soluble Wnt antagonists (sWAs), including soluble frizzled related proteins, dickkopf (Dkk) proteins, and Wnt inhibitory factor-1 (Wif1). Aims: To evaluate the role of sWAs in upper (gastric) and lower (colon) gastrointestinal tract tumorigenesis. Methods: Dkk1-3, Wif1, and FrzB expression was evaluated by in situ RNA hybridisation on normal and malignant human gastric and colon tissues. Expression was graded semiquantitatively. Results: Wif1, Dkk1, and Dkk2 were not expressed in normal gastric tissue. Dkk3 was expressed in some samples, with stronger expression in deep gastric glands. FrzB was expressed in several normal gastric samples, but not in matched tumour specimens. In contrast, Dkk1 and FrzB were not expressed in normal colon. Wif1 was expressed in most colon samples, with stronger expression at crypt bases. Dkk3 and Dkk2 expression was also concentrated at crypt bases. There were no differences between sWA expression in malignant colon and matched normal tissue. Conclusions: sWA expression differed between upper and lower gastrointestinal tract. The loss of FrzB in gastric cancer suggests that it acts as a tumour suppressor. The graded expression of Dkk3 in gastric tissue, and Dkk2, Dkk3, and Wif1 in colon tissue, with increased expression in the deep gastric glands/colonic crypt bases, where gastrointestinal stem cells reside, suggests that sWAs may be crucial Wnt signalling regulators in these tissues, and may contribute to stem cell pool maintenance. sWAs are important components of the gastrointestinal proliferative regulatory network.

Recombinant Chinese hamster ovary (CHO) parental clones expressing a humanized antibody against S surface antigen of hepatitis B virus were obtained by cotransfection of heavy chain (HC) and light chain (LC) cDNA expression vectors into dihydrofolate reductase (DHFR)-deficient CHO cells. When 23 representative parental clones were subjected to stepwise selection for increasing methotrexate (MTX) resistance, such as 0.02, 0.08, 0.32, and 1.0 microM, their clonal variations in regard to antibody expression were found to be significant. Among 23 parental clones, only one clone (hu17) showed the significant increment of specific antibody productivity (q(Ab)) with increasing MTX concentration up to 0.32 microM. Compared with the parental clone (hu17), the q(Ab) of hu17 resistant at 0.32 microM MTX (hu17-0.32) was enhanced approximately 12.5-fold. To clarify the reason for the occurrence of clonal variations, Southern blot analyses of chromosomal DNAs derived from each amplified clone at 0.32 microM MTX were performed. Only the hu17-0.32 clone did not experience severe genetic rearrangement during gene amplification, and it had only one 49-kb amplification unit including the LC and HC cDNAs. A fluorescent MTX competition assay showed that the resistance against MTX toxicity of the other clones without enhanced q(Ab) at 0.32 microM MTX was obtained by mechanisms such as an impaired MTX transport system. Taken together, the data obtained here show that clonal variations in regard to antibody expression are found to be significant because clones can acquire MTX resistance by mechanisms other than DHFR-mediated gene amplification despite the stepwise selection.

Sodium butyrate (NaBu) is used as a productivity enhancer for the production of therapeutic recombinant proteins in Chinese hamster ovary (CHO) cells. However, NaBu is well-known for having a cytotoxic effect, thereby inducing apoptosis. As an endeavor to reduce this defect, we studied 11 antioxidants known for inhibiting apoptosis, according to a Plackett-Burman statistical design on CHO cells producing recombinant interferon-beta-1a (IFN-beta). None of the antioxidants that we tested were as effective as N-acetylcystein (NAC) from the point of view of maintaining long-term survival of CHO cells and increasing the production of IFN-beta. In 7.5-L perfusion bioreactor cultures, the addition of NaBu and NAC elongated the culture period to almost 200 h throughout production phase and increased the production yield by 2-fold compared to control cultures containing only NaBu. Glycosylation patterns of produced IFN-beta at each run were also compared in IEF analysis. IEF profiles of where NaBu and NAC were added showed to be more isoforms with a lower pI than those of the control run. The sialic acid content was also increased by 17.7% according to HPLC analysis. Taken together, the data obtained demonstrate that the addition of NAC has positive effects on the elongation of the culture period, improving the production and increasing the sialylation of IFN-beta in NaBu-treated CHO cells.

The surface plasmon resonance (SPR) biosensor measures the real-time kinetics of noncovalent interaction between a receptor and its ligand. Monoclonal antibodies (mAbs) against recombinant factor VIII (rFVIII) were screened from 127 mAb candidates using the SPR biosensor for the purpose of affinity purification of rFVIII. Each mAb showed a different association and dissociation capacity for rFVIII at each buffer condition. One mAb, F8-38, was selected for immunopurification of rFVIII. To characterize the selected mAb F8-38, the immunopurification results on the anti-FVIII mAb F8-38 affinity gel and the anti-von Willebrand factor (vWF) mAb affinity gel were studied. Immunopurification by the anti-vWF affinity gel showed a lower binding capacity of rFVIII and resulted in low purification efficiency. On the other hand, immunopurification by the anti-FVIII affinity gel exhibited a 3.5-fold binding capacity and a 2-fold purification efficiency compared to those of the anti-vWF affinity gel. The amounts of proteins and DNAs derived from host cells and mouse IgGs derived from the affinity matrix in the affinity eluate were similar to those of the anti-vWF affinity gel. In conclusion, the SPR method of immunopurification is a useful technology in the screening of mAbs aimed at the development of an affinity purification procedure, and the mAb F8-38 was selected using this technology on the basis of the purification procedure of rFVIII.