Expression and characterization of glycolipid-anchored B7-1 (CD80) from baculovirus-infected insect cells: protein transfer onto tumor cells.

Tumor cells can be modified to express immunostimulatory molecules such as B7-1 by protein transfer using purified glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1). In this study recombinant baculovirus encoding GPI-B7-1 (vBacB7-1(GPI)) was established to obtain large quantities of purified GPI-B7-1 to modify tumor cells by protein transfer. vBacB7-1(GPI)-infected insect cells showed high-level cell surface expression of GPI-B7-1 that was susceptible to PIPLC treatment. GPI-B7-1 expressed in insect cells (Bac-GPI-B7-1) mediated T cell proliferation, indicating that the GPI-B7-1 retains costimulatory activity. Moreover, Bac-GPI-B7-1 was completely solubilized in Triton X-100 at 4 degrees C compared to 22% solubilization of GPI-B7-1 expressed in CHOK1 cells, suggesting that GPI-anchored proteins expressed in insect cells may not be clustered into the detergent-insoluble fraction. SDS-PAGE analysis of Bac-GPI-B7-1 showed faster mobility (45 kDa) compared to GPI-B7-1 from CHOK1 (68 kDa) and this difference may be due to a difference in glycosylation. Cell binding assays showed that immunoaffinity-purified Bac-GPI-B7-1 retained its functional ability to bind CD28(+) cells. Moreover, when human tumor cells were incubated with this functionally active purified GPI-B7-1, an efficient transfer of B7-1 onto tumor cells was observed. These results demonstrate that GPI-B7-1 can be expressed in insect cells in a functionally active form and can be used to modify tumor cells for immunotherapeutic applications.     

Release and recovery of porcine pepsin and bovine chymosin from reverse micelles: a new technique based on isopropyl alcohol addition.

After complete solubilization by the direct method, porcine pepsin was not released from AOT in isooctane reverse micelles even under aqueous-phase conditions which would not ordinarily allow uptake. Similarly, bovine chymosin, once forward-transferred at a pH below its isoelectric point, was not back-transferred into an aqueous contact phase buffered at a pH value above its isoelectric point. These results show that there is significant hysteresis in the forward- and backward-transfer processes and further imply that kinetics, and not equilibrium, control uptake or release processes for these enzymes. The addition of 10-15% isopropyl alcohol to the aqueous phase increases the rate of protein release dramatically and allows for nearly complete back-transfer of porcine pepsin and 70% back-transfer of bovine chymosin. IPA addition does not destroy the functional integrity of the system since forward transfer of bovine chymosin still occurs at pH values below (but not above) the pI of the protein.     

Influence of low irradiance on chloroplast proteins and photosystem activities in rice cultivars

In six rice cultivars the relative amounts of 55, 26–28 and 15 kDa polypeptides were mostly lower in plants grown for 15 d under low irradiance than in plants grown under saturating irradiance. This decline in the polypeptides, especially in those related to light-harvesting chlorophyll proteins, was accompanied with a decrease in the whole chain electron transport and the photosystem 2 activity.     

Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis

Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5′ untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.