Organization of higher-level chromatin structures (chromomere,chromonema and chromatin block) examined using visible light-induced chromatin photo-stabilization

The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei.     

Urea-induced denaturation of human serum albumin labeled with acrylodan

We induced the denaturation of unlabeled human serum albumin (HSA) and of similar albumin labeled with acrylodan (6-acryloyl-2-dimethylamino naphthalene) with urea and studied the transition profiles using circular dichroism and fluorescence spectroscopy. The circular dichroism spectra for both albumin preparations resulted in the same curves, thus indicating that labeling with acrylodan does not perturb the conformation of HSA. Our results indicate that the denaturation of both albumin preparations takes place at a single, two-state transition with midpoint at about 6 M urea, due to the unfolding of its domain II. It is important to point out that even at 8 M urea, some residual structure remains in the HSA. Great changes in the fluorescence of the dye bound to the protein were observed by addition of solid guanidine hydrochloride to the protein labeled with acrylodan dissolved in 8 M urea, indicating that domain I of this protein was not denatured by urea.     

The SU Glycoprotein 120 from HIV-1 Penetrates into Lipid Monolayers Mimicking Plasma Membranes

Increasing evidence suggests that the HIV envelope binds through its surface (SU) gp120 not only to receptors and coreceptors, but also to other components of the cellular membrane where the glycolipids appear to be good candidates. To assess the ability of HIV-1 SU gp120 to penetrate into phospholipid membranes, we carried out a study of the interactions between a recombinant SU gp120 from HIV-1/HXB2 and artificial lipid monolayers mimicking the composition of the outer leaflet of the lymphocytes and which were spread at the air-water interface. We show that the protein, in its aggregated form, has amphipathic properties and that the insertion of this amphipathic species into lipids is favored by the presence of sphingomyelin. Furthermore, cholesterol enhances the penetration into mixed phosphatidylcholine-sphingomyelin monolayers. Coexistence of different physical states of the lipids and thus of domains appears to play a major role for protein penetration independently of the presence of receptors and coreceptors. Received: 24 April 2000/Revised: 11 July 2000     

New approaches to the study of sphingolipid enriched membrane domains: The use of microscopic autoradiography to reveal metabolically tritium labeled sphingolipids in cell cultures

This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture.To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3×10^–7 M [1-³H]sphingosine. [1-³H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-³H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy.Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.     

A Theoretical Model for the Association Probabilities of Saturated Phospholipids from Two-Component Bilayer Lipid Membranes

The non-random mixing of biomembrane components, especially saturated phospholipids, exhibits important consequences in molecular biology. Particularly, the distribution of lipids within natural and model membranes is strongly determined by the selective association processes. These processes of phospholipids take place due to the cooperative modes in multiparticle systems as well as the specific lipid-lipid interactions both in the hydrophobic core and in the region of the polar headgroups. We demonstrated that the investigation of the selective association processes of saturated phospholipids might contribute to the insight of the lipid domains appearance inside the bilayer membranes. The association probabilities of like-pairs and cross-pairs from a binary mixture of saturated phospholipids were tested for both parallel and anti-parallel alignments of the polar headgroups. The present model confirms the experimental evidence for saturated phospholipids to have a high tendency for association in parallel configuration of the electric dipole moments of the polar headgroups whether the cross-sectional area of the polar headgroup is in an usual range of 25-55 2. There are three major lipid domains in a binary mixture of saturated phospholipids: (i) lipid domains in non-mixed phase of the first mixture component, in parallel alignment of the polar headgroups; (ii) lipid domains in non-mixed phase of the second mixture component, in anti-parallel alignment of the polar headgroups; (iii) lipid domains in mixed phase. We think that the selective association processes of phospholipids are neither exclusively, nor only involved in promoting the lipid domains appearance through bilayer phospholipid membranes.     

Division polarity and plasticity of the meiosis I spindle inCypripedium californicum (Orchidaceae)

Summary Establishment of division polarity and meiotic spindle organization in the lady's slipper orchidCypripedium californicum A. Gray was studied by immunocytochemistry, confocal and transmission electron microscopy. Prior to organization of the spindle for meiosis I, the cytoplasmic domains of the future dyad and spindle polarity are marked by: (1) constriction of the prophase nucleus into an hourglass shape; (2) reorganization of nuclear-based radial microtubules into two arrays that intersect at the constriction; and (3) redistribution of organelles into a ring at the boundary of the newly defined dyad domains. It is not certain whether the opposing microtubule arrays contribute directly to the anastral spindle which is organized in the perinuclear areas of the two hemispheres. By late prophase each half-spindle consists of a spline-like structure from which depart the kinetochore fibers. This peculiar spindle closely resembles the spline-like spindle of generative-cell mitosis in certain plants where the spindle is distorted by physical constraints of the slender pollen tube. In the microsporocyte, the elongate spindle of late prophase/metaphase is curved within the cell so that the poles are not actually opposite each other and chromosomes do not form a plate at the equator. By late telophase the poles of the shortened halfspindles lie opposite each other. Plasticity of the physically constrained plant spindle appears to be due to its construction from multiple units terminating in minipoles. Cytokinesis does not follow the first meiosis. However, the dyad domains are clearly defined by radial microtubules emanating from the two daughter nuclei and the domains themselves are separated by a disc-like band of organelles.     

Structural characterization of the entire 1.3S subunit of transcarboxylase from Propionibacterium shermanii.

Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123-amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with uniform 15N labeling. The backbone structure and dynamics of the protein have been characterized in aqueous solution by three-dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear 3J(HNHalpha) coupling constants, and amide proton exchange data. The protein contains a predominantly disordered N-terminal half, while the C-terminal half is folded into a compact domain comprising eight beta-strands connected by short loops and turns. The topology of the C-terminal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26-30% sequence similarity.     

Universal nuclear domains of somatic and germ cells: some lessons from oocyte interchromatin granule cluster and Cajal body structure and molecular composition

It is now clear that two prominent nuclear domains, interchromatin granule clusters (IGCs) and Cajal bodies (CBs), contribute to the highly ordered organization of the extrachromosomal space of the cell nucleus. These functional domains represent structurally stable but highly dynamic nuclear organelles enriched in factors that are required for different nuclear activities, especially RNA biogenesis. IGCs are considered to be the main sites for storage, assembly, and/or recycling of the essential spliceosome components. CBs are involved in the biogenesis of several classes of small RNPs as well as the modification of newly assembled small nuclear RNA. We have summarized data on the molecular composition, structure, and functional roles of IGCs and CBs in the nuclei of mammalian somatic cells and oocytes of some animals with a special focus on insects. We have focused on similarities and differences between the IGCs and CBs of oocytes and the well‐studied CBs and IGCs of cultured mammalian somatic cells. We have shown the heterogeneous character of oocyte IGCs and CBs, both in structure and molecular content. We have also demonstrated the unique capacity of oocytes to form close structural interactions between IGC and CB components. We proposed to consider these joint structures as integrated entities, sharing the features of both IGCs and CBs.     

Modulation of CD6 function through interaction with Galectin-1 and -3

CD6 is a lymphocyte glycoprotein receptor that physically associates with the antigen-specific receptor complex at the center of the immunological synapse, where it interacts with its ligand CD166/ALCAM. The present work reports the carbohydrate-dependent interaction of CD6 and CD166/ALCAM with Galectin-1 and -3, two well-known soluble mammalian lectins. Both galectins interfered with superantigen-induced T cell proliferation and cell adhesion phenomena mediated by the CD6-CD166/ALCAM pair, while CD6 expression protected cells from galectin-induced apoptosis. The results suggest that interaction of Galectin-1 and -3 with CD6 and CD166/ALCAM might modulate some relevant aspects of T cell physiology.