On the spatial structure of a plant cell wall protein. Secondary structure of a cell wall protein fromAcetabularia

Summary The structure of a purified protein associated with the cell wall polysaccharides of the marine green algaeAcetabularia (Polyphysa) cliftonii has been studied by means of X-ray diffraction, infrared spectroscopy and circular dichroism. The homogeneous preparation of the cell wall protein has a molecular weight of 14,000, as determined by sodium-dodecylsulfate electrophoresis. Regular layer line reflections on the X-ray diffraction photographs suggest that a distinct order exists in the arrangement of the protein fibrils. Through infrared spectroscopy of thin aqueous films of the protein, as well as of the fibers, it was established that the -helical structure is predominant in the cell wall protein. The fibers crystallize in a hexagonal unit cell witha=14.5 Å and c=27.0 Å, at a water content of two molecules per residue. Increase in water content causes an increase in thea-axis, but without change in thec-direction, thus keeping the -helical conformation. Moreover the spectral data in the amide A, I, II, III, and IV-regions show that the cell wall protein has an ordered -helical conformation.     

The fine structure of the human endometrial ciliated cell.

Sixty-eight histologically normal specimens from all stages of the menstrual cycle were examined during an ultrastructural study of the human endometrial ciliated cell. The 'clear' ciliated cells found initially were seen to become at first dense and then secretory. These changes are advanced as an explanation to account for the previously observed reduction in frequency of the ciliated cell during the luteal phase. The possibility of an hormonal control mechanism is discussed.     

Vegetative cell structure in Anabaenopsis sp.

Summary A study was made of the morphology and ultrastructure of Anabaenopsis sp. With the aid of serial sections the 3 dimensional appearance of the cell was studied. The lamellae form an anastomosing network, but isolated pockets of cytoplasm completely surrounded by lamellae probably do not occur. Interesting structures in this organism include clusters of bodies about 30 m in diameter which lie between the plasma membrane and the I layer of the wall, and a well developed system of plasmadesm-like fibrils running between plasma membranes of adjacent cells across both I and M wall layers.     

Fine structure of the cell envelope layers of Flexibacter polymorphus.

Electron microscopy of the filamentous gliding marine bacterium Flexibacter polymorphus demonstrated that the cell envelope consists of an electron-dense intermediate layer located between two unit-type membranes: an outer membrane, presumably of lipopolysaccharide, and an inner cytoplasmic membrane. Separation of living filaments into single cells by lysozyme suggests that a peptidoglycan moiety, possibly corresponding to the intermediate layer, might be situated between the two membranes. Cell division proceeds by invagination of the cytoplasmic membrane and intermediate layer forming a transverse septum. Cells generally fail to separate after the division process, so that a common outer membrane encloses all of the cells in a single filament. There is a continuous layer of macromolecular cup-shaped elements ('goblets') attached to the outermost surface of the lipopolysaccharide membrane. Tangential thin sections, as well as negatively stained preparations of envelope fragments (produced by sonication of autolyzed cells), showed that the goblets are arranged in a close-packed hexagonal array. The presence of electron-dense structures located between the outer and inner membranes, and exhibiting the same periodicity as the goblets, suggests that some part of the goblets penetrates the outer membrane and extends across the periplasmic space to the dense intermediate layer or cytoplasmic membrane. Spontaneous autolysis in aging cultures is accompanied by the formation and release into the culture medium of large numbers of outer membrane vesicles coated with globlets. A tentative reconstruction of the envelope of F. polymorphus, based on the fine-structural data, is presented.     

On the structure of agglutinated sheep red blood cell membranes.

Specimens of isolated sheep red blood cell membranes are prepared by an agglutination technique in which membranes are stacked in regular arrays. X-ray diffraction patterns are recorded from such specimens which show meridional and equatorial diffraction phenomena. The meridional reflections correspond to single lamellar repeat periods of 160-186 A. It is concluded that two asymmetric membranes are contained in the elementary period. Lipid phases with preferentially oriented hydrocarbon chains are part of the membrane structure. The stacking of the membranes is also demonstrated in the electron microscope. The X-ray scattering curve of intracellular hemoglobin of intact sheep red blood cells is recorded to a spacing of about 8 A-1. The broad diffraction rings of this scattering curve are replaced by a series of rather sharp rings, when the red blood cells are agglutinated and placed in a hypertonic medium. Both the presence of a functioning membrane and the agglutination appear to be essential for the full expression of this phenomenon.     

Quaternary structure and assembly process of the T cell receptor.

The human immune system contains T and B lymphocytes which respond in an antigen-specific manner to foreign antigens. These foreign antigens are recognized by multimeric receptors expressed on the cell surface of T and B lymphocytes. The subunits that make up the T and B cell receptor complexes have been identified, but their stoichiometries and positions in the complex remain to be resolved. Elucidation of the quaternary structures is necessary to understand the molecular basis of signal transduction events which follow antigen recognition and will contribute to the design of drugs that can modulate T and B cell responses. Here, I will discuss recent insights into the quaternary structures of the TCR and BCR and the striking similarities between the two, both in the structures of the subunits and in the overall quaternary structures. In addition, the intracellular assembly processes of these receptor complexes will be discussed, as well as the recent realization that these processes appear to be mediated by house-keeping proteins that transiently bind to partial TCR and BCR complexes. Similar to the role of BiP which mediates assembly processes of B cell immunoglobulins, a recently identified intracellular protein of 90 kD, called IP90, appears to play a role in TCR and BCR assembly processes. Analyses of the IP90 protein might contribute not only insight into the folding and assembly processes in lymphocytes, but also into those of newly synthesized proteins in many different cell types.     

Fine structure of the cell walls of Polyporus myllitae Cke. et Mass.

Following a brief survey of the wall structure of the vegetative hyphae of a number of basidiomyeetes, attention is focused upon Polyporus myllitae Cke. et Mass. After removal of the outer amorphous layer by various chemical treatments, the underlying surface is seen to consist of an interwoven network of microfibrils. There is no evidence of any preferred angle of orientation. However, on what is believed to be the inner surface of the hyphal wall, microfibrils show a strong tendency towards a transverse orientation. The resulting structure is compatible with the multi-net concept of cell wall growth of Houwink and Koelofsen. There is no obvious change in microfibril orientation in passing along a hypha towards its tip. Electron-opaque cross-walls partition hyphae, sometimes separate a branch from a parent hypha, and occur in clamp connections. The cross-wall consists of microfibrils underlying, or embedded in, an amorphous matrix. They are circularly arranged around a single central pore which has a thickened rim.     

Evolution of the fibronectin gene. Exon structure of cell attachment domain

Genomic DNA coding for human fibronectin was identified from a human genomic library by screening with a cDNA clone that specifies the cell attachment domain in human fibronectin. Two clones which together provided more than 22 kilobase pairs of the fibronectin gene were isolated. The exons in this region correspond to approximately 40% of the coding region in the fibronectin gene. They code for the middle region of the polypeptide which consists of homologous repeating segments of about 90 amino acids called type III homologies. Nucleotide sequence of the portion of the gene corresponding to the cell attachment domain showed that the Arg-Gly-Asp-Ser cell attachment site is encoded within a 165-base pair exon. This exon, together with a 117-base pair exon codes for a homology unit. Analysis of the exon/intron organization in some of the neighboring homology units indicated a similar 2-exon structure. An exception to this pattern is that a single large exon codes for a type III homology unit that, due to alternative mRNA splicing, exists in some but not all fibronectin polypeptides. The introns separating the coding sequences for the type III homology units are located in conserved positions whereas the introns that interrupt the coding sequence within the units are in a variable position generating variations in the size of the homologous exons. This exon/intron organization suggests that the type III homology region of the fibronectin gene has evolved by a series of gene duplications of a primordial gene consisting of two exons. Specification of one of these homology units to the cell attachment domain has occurred within this exon/intron arrangement.     

Chromatin structure through the cell cycle. Studies with regeneration rat liver.

Liver nuclei were prepared through the first cell cycle in partially hepatectomized young rats showing 30% parenchymal cell synchrony. To determine if nucleosome structure altered during this period, liver nuclei from sham-operated rats were compared with nuclei isolated at various times after partial hepatectomy. These nuclei were exposed to deoxyribonuclease I (EC 3.1.4.5), deoxyribonuclease II (EC 3.1.4.6) or micrococcal nuclease (EC 3.1.4.7) and the nucleosome-associated DNA length was ascertained. In no case was a difference in the DNA lengths associated with nucleosome structure observed. Differences were observed with regard to the histones and their relative association with nuclear material. When nuclei from normal rat livers were incubated in hypo-osmolar medium 9% of histone 1 and 4% of the other histones were released. These released histones, unlike those remaining bound to the nuclei, showed high [3H]adenosine and [3H]acetate uptakes in vivo. [32P]P1 uptake was also much greater into released than bound histones 1 and 3, but was not different for histone2A. At 3.5-4.5 h after partial hepatectomy, the release of histone 1 was trebled and that of histone 4 doubled. By 13.5 h, when phosphorylation of the bound forms of histones 2A and especially 1 was increased, no further changes in histone release in hypo-osmolar medium were found. The released histones from partially hepatectomized livers had indistinguishable [3H]adenosine uptakes from controls. The roles are discussed of phosphorylation and ADP-ribosylation in labilizing histone binding.     

The structure of endothelial cell thrombospondin. Characterization of the heparin-binding domains

The glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet-sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin-binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five-fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme-to-substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30-kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin-cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin-susceptible site, which has been observed in the long connecting arm, which is particularly Ca-stabilized. Heparin-binding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low-affinity binding of the large fragments but not the high-affinity binding of the small fragments. Two-dimensional gel electrophoresis of the chymotryptic heparin-binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin-binding capacity of both high and low-affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)     

A role for water in cell structure.

The question of a role for water in biochemical and cellular events is ignored by most workers (apart from its obvious role in hydrolysis reactions, which is not under discussion here). But much recent research has pointed to the importance of physical, as well as biochemical, processes of the cell, which focus attention on such straightforward elementary questions as position and relationship in space of cell components. In this communication these questions are examined in terms of a new model of water structure. A radically new feature of this model is that water clusters have long-term rather than flickering existence and are as large as the macromolecular components of the cell. These properties allow the clusters and other components to pack together spatially so giving rise to integrated, large-scale, subcellular structures.