Activation of teratocarcinoma-derived hemoglobin genes in teratocarcinoma-Friend cell hybrids.

Hybrid cells formed by the fusion of murine teratocarcinoma and Friend erythroleukemia cells synthesize hemoglobin in the presence of chemical inducers such as dimethylsulfoxide (DMSO). By making use of the fact that the parental teratocarcinoma and Friend cells carried different alleles at the locus coding for the alpha chain of hemoglobin, it was possible to demonstrate that the teratocarcinoma-derived genes for the globin alpha chains are genetically active in hemoglobin-synthesizing hybrid cells. In addition, evidence is presented suggesting that the teratocarcinoma-derived genes for the beta-globin chains may also be expressed in the hybrids. Apparently the teratocarcinoma-derived genome has become reprogrammed to express erythroid functions following fusion of the teratocarcinoma cell to the Friend cell.     

H－ras在颊粘膜正常、良性、癌前及恶性变上皮中的表达：免疫组织化学定量研究

本文通过建立图象分析方法对免疫组织化学反应结果进行定量,检测观察Ｈ－ｒａｓ在口腔颊粘膜上皮在正常（Ｎ）、慢性炎症（ＩＦ）、癌旁上皮（ＥＡＣ）和鳞癌（ＳＣＣ）的变化过程中的表达并进行分析。结果显示Ｈ－ｒａｓ在ＳＣＣ组中,以中等分化的ＳＣＣ无论是Ｈ－ｒａｓ表达的量还是细胞阳性率都较高。此外,组织学观察显示,Ｈ－ｒａｓ在处于分化末期但尚未角化的正常上皮细胞中有较高的表达。本文结果显示了Ｈ－ｒａｓ的过表达与上皮细胞的会化程度密切相关。本研究还显示,所采用的阳性区域透光值、平均总透光值及阳性反应区域与阴性反应区域比值可靠并有相关性。这进一步说明了用免疫组化定量方法检测Ｈ－ｒａｓ癌基因表达的精确和可靠性。     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

The isolation and physiological analysis of mutants of the moss,Physcomitrella patens,which over-produce gametophores

We have compared the effects of cycloheximide (CHI) and two other rapid and effective inhibitors of protein synthesis, pactamycin and 2-(4-methyl-2,6-dinitroanilino)-N-methyl proprionamide (MDMP), on protein synthesis, respiration, auxin-induced growth and H⁺-excreation of Avena sativa L. coleoptiles. All three compounds inhibit protein synthesis without affecting respiration. The effectiveness of the inhibitors against H⁺-excretion and growth correlates with their ability to inhibit protein synthesis. Both CHI and MDMP inhibit auxin-induced H⁺-excretion after a latent period of 5–8 min, and inhibit growth after a 8–10-min lag. These results support the idea that continued protein synthesis is required in the initial stages of the growth-promoting action of auxin.Abbreviations CHI cycloheximide - DMSO dimethyl sulfoxide - FC fusicoccin - IAA indole-3-acetic acid - MDMP 2-(4-methyl-2,6-dinitroanilino)-N-methyl proprionamide     

Expression of globin genes in teratocarcinoma-Friend cell hybrids

We have used an isoelectric focusing technique to analyse the hemoglobins synthesized by cell hybrids isolated following the fusion of Friend erythroleukemia to embryonal carcinoma cells. Our results confirm that the embryonal carcinoma-derived alpha globin genes are expressed in cell hybrids. In addition, the extent to which the various alpha chains and beta chains are synthesized in these hybrids depends on both the genetic composition of the cell line and on the chemical nature of the agent used to induce hemoglobin synthesis.     

Lock and key binding of the HOX YPWM peptide to the PBX homeodomain

HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.     

A conformational change in PBX1A is necessary for its nuclear localization

The fly homeodomain (HD) protein EXTRADENTICLE (EXD) is dependent on a second HD protein, HOMOTHORAX (HTH), for nuclear localization. We show here that in insect cells the mammalian homolog of EXD, PBX1A, shows a similar dependence on the HTH homologs MEIS1, 2, and 3 and the MEIS-like protein PREP1. Paradoxically, removal of residues N-terminal to the PBX1A HD abolishes interactions with MEIS/PREP but allows nuclear accumulation of PBX1A. We use deletion mapping and fusion to green fluorescent protein to map two cooperative nuclear localization signals (NLSs) in the PBX HD. The results of DNA-binding assays and pull-down experiments are consistent with a model whereby the PBX N-terminus binds to the HD and masks the two NLSs. In support of the model, a mutation in the PBX HD that disrupts contact with the N-terminus leads to constitutive nuclear localization. The HD mutation also increases sensitivity to protease digestion, consistent with a change in conformation. We propose that MEIS family proteins induce a conformational change in PBX that unmasks the NLS, leading to nuclear localization and increased DNA-binding activity. Consistent with this, PBX1 is nuclear only where Meis1 is expressed in the mouse limb bud.