Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2＇-deoxycytidine

The non-classical HLA class Ⅰ antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells （DC）. As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2＇-deoxycytidine （5-AC） and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5＇ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components （APM） as well as β2 microglobulin （β2-m） expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.     

Human embryonic stem cell lines derived from the Chinese population

Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.     

Recoverin improves rod-mediated vision by enhancing signal transmission in the mouse retina

Vision in dim light requires that photons absorbed by rod photoreceptors evoke signals that reliably propagate through the retina. We investigated how a perturbation in rod physiology affects propagation of those signals in the retina and ultimately visual sensitivity. Recoverin is a protein in rods that prolongs phototransduction and enhances visual sensitivity. It is not present in neurons postsynaptic to rods, yet we found that light-evoked responses of rod bipolar and ganglion cells were shortened when measured in recoverin-deficient retinas. Unexpectedly, the effect of recoverin on postsynaptic signals could not be explained by its effect on phototransduction. Instead, it is an effect of recoverin downstream of phototransduction in rods that prolongs signal transmission and enhances visual sensitivity. An important implication of our findings is that the recovery phase of the rod photoresponse does not contribute significantly to visual sensitivity near absolute threshold.     

Protection against malaria by anti-erythropoietin antibody due to suppression of erythropoiesis in the liver and at other sites

We have previously reported that erythropoiesis commences in the liver and spleen after malarial infection, and that newly generated erythrocytes in the liver are essential for infection of malarial parasites as well as continuation of infection. At this time, erythropoietin (EPO) is elevated in the serum. In the present study, we administered EPO or anti-EPO antibody into C57BL/6 (B6) mice to modulate the serum level of EPO. When mice were infected with a non-lethal strain (17NXL) of Plasmodium yoelii (blood-stage infection of 10(4) parasitized erythrocytes per mouse), parasitemia continued for 1 month, showing a peak at day 17. Daily injection of EPO (200 IU/day per mouse) from day five to day 14 prolonged parasitemia, whereas injection of anti-EPO antibody (1.5 mg/day per mouse) every second day from day five to day 28 decreased it. Erythropoiesis was confirmed in the liver, spleen and bone marrow by the appearance of nucleated erythrocytes (TER119+). When anti-EPO antibody was injected by the same protocol into mice infected with a lethal strain (17XL) of P. yoelii, all mice showed decreased parasitemia and recovered from the infection. These results suggest that the use of anti-EPO antibody after malarial infection may be of therapeutic value in severe cases of malaria.     

Stage-dependent gene expression profiles during natural killer cell development

Natural killer (NK) cells develop from hematopoietic stem cells (HSCs) in the bone marrow. To understand the molecular regulation of NK cell development, serial analysis of gene expression (SAGE) was applied to HSCs, NK precursor (pNK) cells, and mature NK cells (mNK) cultured without or with OP9 stromal cells. From 170,464 total individual tags from four SAGE libraries, 35,385 unique genes were identified. A set of genes was expressed in a stage-specific manner: 15 genes in HSCs, 30 genes in pNK cells, and 27 genes in mNK cells. Among them, lipoprotein lipase induced NK cell maturation and cytotoxic activity. Identification of genome-wide profiles of gene expression in different stages of NK cell development affords us a fundamental basis for defining the molecular network during NK cell development.     

Herpes simplex virus type 1 UL51 protein is involved in maturation and egress of virus particles

The UL51 gene of herpes simplex virus type 1 (HSV-1) encodes a phosphoprotein whose homologs are conserved throughout the herpes virus family. Recently, we reported that UL51 protein colocalizes with Golgi marker proteins in transfected cells and that targeting of UL51 protein to the Golgi apparatus depends on palmitoylation of its N-terminal cysteine at position 9 (N. Nozawa, T. Daikoku, T. Koshizuka, Y. Yamauchi, T. Yoshikawa, and Y. Nishiyama, J. Virol. 77:3204-3216, 2003). However, its role in the HSV replication cycle was unknown. Here, we generated UL51-null mutants (FDL51) in HSV-1 to uncover the function of UL51 protein. We show that the mutant plaques were much smaller in size and that maximal titers were reduced nearly 100-fold compared to wild-type virus. Electron microscopy indicated that the formation of nucleocapsids was not affected by the deletion of UL51 but that viral egress from the perinuclear space was severely compromised. In FDL51-infected cells, a large number of enveloped nucleocapsids were observed in the perinuclear space, but enveloped mature virions in the cytoplasm, as well as extracellular mature virions, were rarely detected. These defects were fully rescued by reinsertion of the UL51 gene. These results indicate that UL51 protein is involved in the maturation and egress of HSV-1 virus particles downstream of the initial envelopment step.     

Fe(II)/Cu(I)-dependent P-type ATPase activity in the liver of Long-Evans cinnamon rats

This study examined Fe(II)-dependent ATPase activity in OTG (octylthioglucoside) -treated microsomes isolated from Wistar and LEC rats. The ATPase activity of the liver OTG-microsomes from Wistar rats increased sharply in the 5-150 microM range of Fe(II) with a K0.5 value of 23.9+/-3.6 microM, while the activity of LEC rat liver microsomes increased with increasing Fe(II) up to 500 microM with a K0.5 value of 64.4+/-8.1 microM. The K0.5 values for Fe(II)-dependent ATPase activity of spleen OTG-microsomes were nearly identical at 59.3 microM in the Wistar rat and 63.7 microM in the LEC rats with a similar level of activity at each Fe(II) concentration in both strains of animals. These results indicated that there are two types of Fe(II)-dependent ATPase with different Fe(II) sensitivity, a high sensitive (H) and a low sensitive (L) type, and that the H-type activity was specific to the liver. The H-type activity was, however, deficient in the liver of LEC rats that accumulate copper and iron in hepatocytes as a result of mutations in the Wilson's disease protein (WNDP). On the basis of these results, together with the similarity in optimal conditions required for full activity of the enzyme, we conclude that the Fe(II)-dependent ATPase (H-type) and WNDP may be identical.     

Spectral differentiation of blue opsins between phylogenetically close but ecologically distant goldfish and zebrafish

Zebrafish and goldfish are both diurnal freshwater fish species belonging to the same family, Cyprinidae, but their visual ecological surroundings considerably differ. Zebrafish are surface swimmers in conditions of broad and shortwave-dominated background spectra and goldfish are generalized swimmers whose light environment extends to a depth of elevated short wavelength absorbance with turbidity. The peak absorption spectrum (lambdamax) of the zebrafish blue (SWS2) visual pigment is consistently shifted to short wavelength (416 nm) compared with that of the goldfish SWS2 (443 nm). Among the amino acid differences between the two pigments, only one (alanine in zebrafish and serine in goldfish at residue 94) was previously known to cause a difference in absorption spectrum (14-nm lambdamax shift in newt SWS2). In this study, we reconstructed the ancestral SWS2 pigment of the two species by applying likelihood-based Bayesian statistics and performing site-directed mutagenesis. The reconstituted ancestral photopigment had a lambdamax of 430 nm, indicating that zebrafish and goldfish achieved short wavelength (-14 nm) and long wavelength (+13 nm) spectral shifts, respectively, from the ancestor. Unexpectedly, the S94A mutation resulted in only a -3-nm spectral shift when introduced into the goldfish SWS2 pigment. Nearly half of the long wavelength shift toward the goldfish pigment was achieved instead by T116L (6 nm). The S295C mutation toward zebrafish SWS2 contributed to creating a ridge of absorbance around 400 nm and broadening its spectral sensitivity in the short wavelength direction. These results indicate that the evolutionary engineering approach is very effective in deciphering the process of functional divergence of visual pigments.     

Intracellular signal-responsive gene carrier for cell-specific gene expression

We designed a peptide-polymer conjugate (CPCCtat) as a novel gene carrier that could control gene expression responding to the intracellular caspase-3 signal. This carrier consists of an uncharged main polymer chain and a cationic peptide side chain, which includes the substrate sequence of caspase-3 and the protein transduction domain sequence of HIV-1 Tat. In the present study, CPCCtat formed a tight complex with DNA through an electrostatic interaction, and in this state the gene expression was totally suppressed. In contrast, the complex disintegrated in the presence of caspase-3 due to cleavage of the cationic portion from CPCCtat. This event led to an activation of gene expression. Our results also indicate that the complex can be delivered into living cells due to the cell-permeable peptide side chain of CPCCtat. This intracellular signal-responsive system with CPCCtat will be useful for the cell-specific gene expression system.