Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast.

We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with chymosin in yeast. From this library, we obtained two VHH clones that stimulated chymosin secretion by screening colonies for the level of chymosin secreted. These VHHs bound biotinylated chymosin in an immunoblot procedure but failed to bind chymosin in ELISA, suggesting that their interaction with chymosin was of low affinity. In a second approach, chymosin-specific VHHs were first selected using phage display and then coexpressed with chymosin in yeast cells. Screening yeast cells for higher levels of chymosin secretion resulted in 11 VHHs. Sequence analysis revealed that these 11 VHHs formed four sets of related VHHs that were different from the previously isolated two VHHs. Although binding of VHHs to chymosin could not be demonstrated in ELISA using soluble VHHs, it could be unambiguously demonstrated for clones isolated by phage display, using phage-displayed VHHs. Finally, quantitative Western blot analysis of chymosin amounts demonstrated that coexpression with VHH domains can stimulate the level of secreted chymosin 1.5- to 6-fold.

Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen.

Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection.

Wavelength-dependent effects of benzoporphyrin derivative monoacid ring A in vivo and in vitro.

Benzoporphyrin derivative monoacid ring A (BPD-MA) is a chlorin-like photosensitizer currently in clinical trials for cancer and psoriasis. It has maximal absorption peaks at both 630 and 690 nm and can be activated at both these wavelengths. In vitro phototoxicity tests using the P815 murine mastocytoma cell lines conducted over wavelengths of light between 678 and 700 nm emitted by an argon-ion pumped dye laser showed that equivalent cell kill could be achieved between 682 and 690 nm. Tests on in vivo phototoxicity of normal skin of DBA/2 mice injected with 2 mg/kg of BPD-MA and exposed to light at 125 J/cm2, between 620 and 700 nm, demonstrated peaks of normal skin damage occurring at 630-640 nm and 680-690 nm. In tests carried out with light between 620 and 700 nm, at 10 nm increments, it was seen that light delivered at 680-690 nm caused slightly more damage to normal skin than light delivered at 630-640 nm. When lower doses of light between 675 and 705 nm were tested using smaller increments, it was determined that equivalent skin damage occurred over a range of 680-695 nm. Antitumor efficacy in tumor-bearing DBA/2 mice was tested between 683 and 695 nm. It was found that equivalent antitumor efficacy, determined by assessing tumor-free status at 20 days posttreatment, occurred at wavelengths between 685 and 693 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Photofrin, but not benzoporphyrin derivative, stimulates hematopoiesis in the mouse.

This report describes the effects of the porphyrin photosensitizers, Photofrin and benzoporphyrin derivative (BPD) on the immunohematopoietic system of normal and immunosuppressed DBA/2 mice in the absence of activating light. Photofrin (10 and 25 mg/kg) significantly increased in vitro colony formation by cells of the granulocyte-macrophage lineage in the spleen and bone marrow. Splenic hypercellularity, splenomegaly and elevated levels of blood leukocytes were observed in these mice 7 days following Photofrin injection. Evidence that Photofrin influenced the lymphohematopoietic compartment was suggested by a significant increase in blood lymphocytes and a population of spleen cells identified by a monoclonal antibody (LR-1) reactive with mouse splenic B lymphocytes. Proliferative responses of spleen cells from Photofrin-treated mice to sub-optimal concentrations of Con A were greater than that observed for controls. However, spleen cell responses to LPS were unaltered by Photofrin administration. In contrast, BPD (10 mg/kg) did not alter any of the immunohematopoietic parameters studied. When Photofrin was administered to mice treated with the myeloablative agent 5-FU there was a significant acceleration in the recovery of total blood leukocyte and spleen cell numbers, relative to the controls. These studies demonstrate that, in addition to its previously documented activities as a photosensitizer, Photofrin can exert stimulatory effects upon murine hematopoiesis.