Homeotic protein binding sites, origins of replication, and nuclear matrix anchorage sites share the ATTA and ATTTA motifs.

Nuclear matrix organizes the mammalian chromatin into loops. This is achieved by binding of nuclear matrix proteins to characteristic DNA landmarks in introns as well as proximal and distal sites flanking the 5' and 3' ends of genes. Matrix anchorage sites (MARs), origins of replication (ORIs), and homeotic protein binding sites share common DNA sequence motifs. In particular, the ATTA and ATTTA motifs, which constitute the core elements recognized by the homeobox domain from species as divergent as flies and humans, are frequently occurring in the matrix attachment sites of several genes. The human apolipoprotein B 3' MAR and a stretch of the Chinese hamster DHFR gene intron and human HPRT gene intron shown to anchor these genes to the nuclear matrix are mosaics of ATTA and ATTTA motifs. Several origins of replication also share these elements. This observation suggests that homeotic proteins which control the expression level of many genes and pattern formation during development are components of the nuclear matrix. Thus, the nuclear matrix, known as the site of DNA replication, might sculpture the crossroads of the differential activation of origins during development and S-phase and the control of gene expression and pattern formation in embryogenesis.     

Differences in prolyl-leucyl-glycinamide metabolism by hypothalamus,pituitary and cerebral cortex of male and female rats

l-Prolyl-l-leucyl-glycinamide is rapidly hydrolyzed by hypothalamic, hypophyseal and cortical homogenates from male or female rats. The peptidase activity is higher in the pituitary followed in decreasing order by the hypothalamus and the cerebral cortex. It is mostly localized in the supernatant fraction of a 100,000 g centrifugation and is inhibited by bacitracin.Tissues from female rats are half as active as those from male rats and show variations during the estrous cycle, with very low PLG metabolism at diestrus 1 in pituitary and hypothalamus. In contrast, the cerebral cortex at proestrus and estrus has significant lower hydrolyzing activity than at diestrus. No change of the peptidase activity is observed in tissues from ovariectomized animals after treatment with estrogen or progesterone.The results obtained suggest the existence of a correlation between peptidasic activity and melanotropin secretion.     

Toxoplasma gondii: Electron microscopic study on the dye test reaction

When intact Toxoplasma trophozoites were stained with isotonic alkaline methylene blue solution, the organelles rich in nucleic acid, i.e., nucleus, free and membrane-bound ribosomes appeared as electron dense areas. When the parasites were incubated with the anti-Toxoplasma antibody and the accessory factor, swelling of the surface membrane occurred first, followed by destruction of the inner structures. In the dye test positive parasites, there were no definite organelles recognizable, as there were in the intact parasites. By the negative staining method, holes (defects) with dark central portions were observed on the surface of the parasites treated with the antibody and the accessory factor, the diameter of the holes measuring about 10–11 nm. These holes, which tended to occur in clusters, were each surrounded by a clear ring.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

Lipid polarity and sorting in epithelial cells

Apical and basolateral membrane domains of epithelial cell plasma membranes possess unique lipid compositions. The tight junction, the structure separating the two domains, forms a diffusion barrier for membrane components and thereby prevents intermixing of the two sets of lipids. The barrier apparently resides in the outer, exoplasmic leaflet of the plasma membrane bilayer. First data are now available on the generation of these differences in Madin-Darby canine kidney (MDCK) cells, grown on filter supports. Experiments in which fluorescent precursors of apical lipids were introduced into the cell have demonstrated that upon biosynthesis apical lipids are sorted from basolateral lipids in an intracellular compartment. In this paper we present a model for the sorting process, the central point of which is that the two sets of lipids laterally segregate into microdomains that bud to form vesicles delivering the lipids to the apical and the basolateral plasma membrane domains, respectively.     

Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic ∼9 nmF ₁ (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the “etched” surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with the freeze-dried TU particles (trypsin-urea treated SMP) that are devoid ofF ₁ (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-“etching.”     

Influence of retinoids and EGF on growth of embryonic mouse palatal epithelia in culture

Summary Retinoids and growth factors seem to be important for normal mammalian reproduction and development. High levels of retinoic acid are teratogenic and induce cleft palate in the mouse. Little is known concerning the mechanisms through which retinoids induce cleft palate. Palatal epithelia from CD-1 embryonic mice on Day 12 of gestation were isolated from the mesenchyme and cultured in serum-free media, with all-trans retinoic acid or 13-cis retinoic acid, with or without epidermal growth factor (EGF). The epithelia attached and grew, and the cells differentiated over a 72-h culture period. Binding of [¹²⁵I]EGF was observed in all cultures in a pattern that correlated with thymidine (TdR) uptake by the epithelia. EGF enhanced growth and [³H]TdR incorporation of the oral cells, but nasal cells generally did not proliferate. In this culture system, both retinoids suppressed [³H]TdR incorporation in a concentration-dependent manner for epithelia cultured with or without EGF. Medial cells are important to normal palatogenesis as they play a role in fusion of opposing shelves and subsequently many of these cells undergo programmed cell death. Death of medial cells in vitro is prevented by EGF and by the retinoids, either with or without EGF. This response occurs in the absence of a mesenchymal interaction, suggesting that the medial cell response to EGF and retinoids is not mediated by or dependent on the mesenchymal tissues. The survival of medial cells may be responsible for the failure of opposing shelves to fuse.     

Immunocytochemical localization of the elongation factor Tu in E. coli cells

The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labelling technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been suggested that EF-Tu could be part of a putative prokaryotic cytoskeleton, we did not find any evidence for supramolecular assemblies, such as fibres or filaments, containing a large amount of EF-Tu. EF-Tu was not observed in association with the outer cell membrane and periplasmic space. A topological relationship with the inner membrane is not apparent in our micrographs. In cells in which the EF-Tu level is raised significantly, the protein piles up in discrete cell regions.