2-D structure of the Neurospora crassa plasma membrane ATPase as determined by electron cryomicroscopy.

Large, well-ordered 2-D crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H(+)-ATPase grow rapidly on the surface of a polyethylene glycol-containing mixture similar to that originally developed for growing 3-D crystals of this integral membrane transport protein. Negative stain electron microscopy of the crystals shows that many are single layers. Cryoelectron microscopy of unstained specimens indicates that the crystals have a p6 layer group with unit cell dimensions of a = b = 167 A. Image processing of selected electron micrographs has yielded a projection map at 10.3 A resolution. The repeating unit of the ATPase crystals comprises six 100 kDa ATPase monomers arranged in a symmetrical ring. The individual monomers in projection are shaped like a boot. These results provide the first indications of the molecular structure of the H(+)-ATPase molecule. They also establish the feasibility of precipitant-induced surface growth as a rapid, simple alternative to conventional methods for obtaining 2-D crystals of the integral membrane proteins useful for structure analysis.     

The structure and mitotic behaviour of a nuclear inclusion in a fern

Summary A study has been made of the structure and behaviour during mitosis of a crystalline inclusion within cell nuclei of roots of Dryopteris filix-max. The inclusion within the interphase nucleus is an aggregate of randomly oriented crystals. All the crystals are similar, and consist of a cubic array of particles of unit spacing approximately 100 Å. During mitosis, the inclusions are eliminated from the nucleoplasm at prometaphase. The crystals reappear within the nucleus at early interphase by a process of random crystallisation from a preformed mass of amorphous material. The results are discussed in the light of previous work on nuclear inclusions in plants and of current theories of the mode of action of microtubules.     

Adenosine triphosphatase from rat liver mitochondria. Crystallization and x-ray diffraction studies of the F1-component of the enzyme.

The homogeneous rat liver F1-ATPase preparation of Catterall and Pedersen (Catterall, W.A., and Pedersen, P.L. (1971) J. Biol. Chem. 246, 4987-4994) has been crystallized from a solution containing phosphate and ATP by precipitation with ammonium sulfate. Most of the resultant crystals are cubes of approximately 0.3 to 0.6 mm per side. X-ray precession photographs show that the crystals are rhombohedral, space group R32 (D37 NO155) with hexagonal cell dimensions a = 148 A, c = 368 A. The molecular weight of the asymmetric unit of the crystals is 190,000 or about half the molecular weight (384,000) of the rat liver enzyme indicating that the crystallographic 2-fold axes of symmetry coincide with a molecular symmetry axis. The crystals diffract to at least 3.5 A and therefore this is the first report of an ATPase preparation in which crystals suitable for x-ray analysis have been obtained.     

Crystals of an ATPase fragment of bovine clathrin uncoating ATPase

A 44,000 Mr amino-terminal, clathrin-independent ATPase fragment of the bovine clathrin uncoating ATPase has been crystallized in a form suitable for X-ray diffraction studies. The crystals are orthorhombic, space group P2(1)2(1)2(1), a = 145.3 A, b = 65.0 A, c = 46.9 A, with one protein molecule per asymmetric unit (1 A = 0.1 nm).     

Electron Microscopy of Monkey Kidney Cell Cultures Infected with Rubella Virus

Two rubella virus strains isolated in this laboratory were investigated in terms of their growth in LLC-MK(2) cell cultures and their effect on cell morphology. Rubella virus grew readily in LLC-MK(2) cells, but cytopathic effects of the virus were not observed in infected cultures. Such infected cultures can be subcultured indefinitely and continue to shed virus. Examination of rubella-infected cell cultures by electron microscopy showed the presence of annulate lamellae in the cytoplasm of 15% of the cells. No changes were evident in the nuclei. These membranous inclusions varied in complexity from parallel arrays of annulate lamellae to large lamellar structures of complex morphology. An occasional cell contained a crystal lattice structure in association with the lamellae. Larger inclusions, consisting of disorganized arrays of "unit" membranes, were also found. Uninfected cells were devoid of annulate lamellae, crystals, and complex membranous inclusions. No viruslike particles were observed in any part of the cells from infected cultures. The significance of the structures observed has not been determined.     

Preliminary crystallographic analysis of the ATP-hydrolysing domain of the Escherichia coli DNA gyrase B protein.

The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain. This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation. Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da). The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A. The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source. The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da). Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives. Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined. Higher resolution native and derivative data are being collected.     

The liver of the slender salamander Batrachoseps attenuatus

Summary The structure of the crystalline inclusions found in Batrachoseps liver cells is described and it is shown that the most symmetric unit cell upon which the crystal lattice is built is a face-centered cube. Taking into consideration the physical properties of a face-centered cubic structure, an attempt is made to determine the nature of the macromolecules that comprise the crystal. It is concluded on the basis of available evidence that the macromolecules probably represent serum lipoproteins. The intracellular synthesis of the crystals and the possible functions they may subserve in the animal are discussed. A comparison is made between the crystals and granules in rat hepatocytes discussed by Bruni and Porter (1965).Research supported by grant RG-6729 from the Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service.     

A NEW CRYSTAL-CONTAINING CELL IN HUMAN ADRENAL CORTEX

Electron microscope examination of the adrenal cortex from three male human subjects revealed a special type of cell occurring in periendothelial spaces, in all adrenal cortex zones. It is a clear, spindle-shaped cell the principal cytoplasmic features of which are crystalline inclusions with a structure similar to that of the Reinke crystals of human testicular interstitial cells and an abundance of microfilaments. Enzymatic digestions with pronase, pepsin, and ribonuclease were performed, and no digestion of the crystals was obtained. The crystals had no peroxidase or acid phosphatase activities. This cell appears to be exclusive to human males and it may be related to adrenal androgen secretion.     

SEROLOGICAL STUDIES ON THE FORMATION OF PROTEIN PARASPORAL INCLUSIONS IN BACILLUS THURINGIENSIS

A strain of Bacillus thuringiensis has been isolated, and methods have been developed for separation of the crystalline, parasporal inclusions in a pure form. Normal sporulation with concomitant crystal formation takes place when cells are incubated under suitable conditions in a nutrient free medium. Serological techniques have been used to study the origin and development of the crystals. Rabbit antisera have been prepared to a vegetative cell extract, suspensions of crystals, and a solution of crystal protein (obtained by alkali treatment of crystals). Tests have been carried out mainly by the Ouchterlony gel plate technique. Crystal protein solutions were found to be more active than suspensions of intact crystals both in reaction with, and in neutralization of, the crystal antibodies. Antisera to the vegetative cell extract gave no reaction with crystal protein. Ultrasonic extracts of cells taken before or during crystal formation gave no reaction with the crystal antibodies. Tests with alkali extracts of disrupted cells showed that the crystal antigen is absent in vegetative cells but arises during sporulation. The appearance of the antigen can be correlated with the formation and growth of the crystals as followed by examination of disrupted cell preparations under the electron microscope. It can be concluded that the crystalline protein inclusions do not arise from precursors in the same antigenic state.     

Cytochemical Observations on Proteins,Alkaline and Acid Phosphatases,Adenosine Triphosphatase,and 5′-Nucleotidase in Chick Liver Cell Cultures Infected with Trichomonas vaginalis*, †

SYNOPSIS. By means of the ninhydrin-Schiff method for proteins a diffuse reaction as well as one localized in granular inclusions can be shown in the cytoplasm of fibroblasts, epithelial cells, and macrophages in trypsin-dispersed chick liver cell cultures. Nuclei and nucleoli also take the specific stain. A progressive loss of cytoplasmic and nuclear staining occurs in the fibroblasts in cultures infected with a relatively pathogenic strain of T. vaginalis. A loss occurs in epithelial cells in advanced stages of degeneration, but in less damaged cells, while the diffuse reaction disappears, the number and staining intensity of the cytoplasmic inclusions remain unchanged or possibly may increase somewhat. The intensity of the diffuse reaction and the number and size of the characteristic inclusions increase in the active, parasite-free, experimental macrophages, but phagocytes with trichomonads closely applied to their external surfaces and those containing the flagellates within their cytoplasm typically retain only a few weak-staining inclusions. Similar distribution of alkaline and acid phosphatases occurs in preparations treated according to Gomori's and Burstone's methods, except that no nuclear staining is obtained with the latter. Activity of both enzymes is localized primarily in inclusions which are dispersed thruout the cytoplasm of fibroblasts and epithelial cells and tend to accumulate along the cell membranes and around the nuclei. In the course of infection with T. vaginalis there is a progressive loss of alkaline phosphatase from both cell types; however, the acid phosphatase activity increases. In the control macrophages both enzymes are localized in mostly rather large, rounded cytoplasmic inclusions. The number of such inclusions increases in the parasite-free experimental macrophages, but only a few weak-staining granules remainin phagocytes with engulfed trichomonads and in those whose external surfaces are in direct contact with the parasites. The loss of the inclusions is less apparent in macrophages containing degenerated flagellates than in the ones with healthy trichomonads, but regardless of the condition of the parasites, the highest enzymatic activity is found around them. ATPase and 5′-nucleotidase are localized in small granules dispersed thruout the cytoplasm of fibroblasts and epithelial cells. The granules tend to accumulate along the periphery of the cells and around the nuclei. A diffuse cytoplasmic reaction is present in preparations processed for 5′-nucleotidase. Nuclei and nucleoli give positive reactions for both enzymes. In the course of infection with trichomonads, activity of the 2 enzymes declines in both culture cell types. Control macrophages have diffuse cytoplasmic reaction for ATPase and 5′-nucleotidase and these enzymes are localized also in rounded cytoplasmic inclusions. Activity of both enzymes increases in the parasite-free experimental phagocytes, but little if any diffuse staining and only a few characteristic inclusions are left in macrophages with engulfed healthy trichomonads and in those whose external surfaces are invested with the flagellates. The ninhydrin-Schiff-positive inclusions found in the macrophages appear to be the same as some of those which have acid phosphatase activity and may well be identical with the glycolipoprotein bodies noted by us previously. On the grounds of their chemical constitution and behavior it seems likely that the inclusions are lysosomes.     

Ultrastructural Localization of Adenosine Triphosphatase Activity in Cotyledon Cells of Tomato and Its Changes during Chilling Stress

The ultrastructural localization of adenosine tripkosphatase (ATPase) activity in cotyledon cells of tomato was carried out by use of the cytochemical method of lead phosphate precipitation, and the changes in ATPase activity during chilling stress of the tomato seedlings were studied. The following experimental results have been obtained: 1. The ATPase activity in the cotyledon cells of tomato seedlings germinated and grown at 28 ℃. was located at plasmolemma, plasmodesmata, nucleoli and nuclear chromatin chloroplast lamellae, many sites of cell wall, and the surface of cell wall bordering the intercellular spaces and their inclusions. 2. When the tomato seedlings were subjected to chilling treatment for 4 h. at 5 ℃., the ATPase activity in cotyledon cells was indifferent from that of non-chilling treated seedlings. After chilling treatment for 12 h. at 5 ℃., the reaction of ATPase activity at plasmolemma, and in cell wall and intercellular spaces was markedly reduced. though the high activity reaction of ATPase in nuclei and at chloroplast lamellae was still maintained. When the tomato seedlings were subjected to chilling stress for 24 h. at 5℃., the ATPase activity at plasmolemma and in cell wall was almost inactivated, while the ATPase activity in nuclei and at chloroplast lamellae was only slightly lowered. These results indicated that the chilling injury may influence firstly on the ATPase activity of cell surface (plasmolemma and cell wall). 3. The role of intercellular spaces used as the passage of materials and the process and mechanism of chilling injury are discussed.     

Involvement of macroautophagy in the dissolution of neuronal inclusions

Ubiquitinated inclusions are a common feature of many neurodegenerative conditions. We have proposed that, at least in part, such inclusions may be formed due to dysfunction of the proteasome. We have modeled such proteasomal dysfunction by applying pharmacological inhibitors to cultured embryonic rat cortical neurons. This treatment leads to neuronal death and formation of ubiquitin/-synuclein-positive cytoplasmic inclusions. At late time points following proteasomal inhibition such inclusions are no longer discerned. Instead, many neurons accumulate small ubiquitinated aggregates, which may represent remnants of the inclusions. In this work we have examined a potential mechanism for inclusion dissolution. Electron microscopy images showed activation of macroautophagy at late time points after proteasomal inhibition. Labeling with LysoTracker Red, a dye that accumulates in acidic compartments, or immunostaining for the lysosomal enzyme Cathepsin D, showed an increase in globular staining. Cathepsin D co-localized partially with small ubiquitinated aggregates, but not inclusions. Application of an inhibitor of macroautophagy or of the vacuolar ATPase led to an increase in the number of inclusions and a decrease in small aggregates, whereas an activator of autophagy had the opposite effects. There was no significant change in apoptotic death following these manipulations. We conclude that, following proteasomal inhibition of cultured cortical neurons, there is activation of macroautophagy and of the lysosomal pathway. This activation results in dissolution of ubiquitinated inclusions into small aggregates, without directly impacting neuronal cell death. These data further support the idea that in this model inclusions and neuronal cell death are independent processes.     

Crystallization patterns of membrane-bound (Na+ +K+)-ATPase

Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound (Na+ +K+)-ATPase is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric alpha beta-unit of the enzyme protein. In phosphate-induced crystals an (alpha beta) 2-unit occupies one unit cell suggesting the interactions between alpha beta-units can be of importance in the function of the Na+, K+ pump.     

番茄种子及萌发过程中蛋白体超微结构的研究

对番茄种子蛋白体的结构,类型及萌发过程中的变化进行了详细观察,未萌发种子蛋白体周围由一单层膜包裹,内部为蛋白质基质及分布其中的内含物３部分组成,根据内含物形态和性质上的不同可分为球状晶体,拟晶体和簇晶体３种形式,根据蛋白体所含内含物的不同,将蛋白体划分为５种基本类型：（１）基质蛋白体;（２）球状晶体蛋白体;（３）拟晶体蛋白体;（４）簇晶体蛋白体;（５）复合蛋白体。萌发过程中,蛋白体逐渐降解并液泡化     

Iron containing nuclear inclusions in human pigmentary cirrhosis (author's transl)]

In human pigmentary cirrhosis nuclear (pseudo-)inclusions of cytoplasmic material, containing less or more degenerated and therefore faintly stained hemosiderin granules, are to be observed. But sometimes there are also finely fibrillar or granular proteinaceous materials, stainable by the Prussian-blue reaction, lying between the chromatin-strands or occupying the whole nucleus and displacing the chromatin to the nuclear envelope (margination of chromatin). Such uncoloured substances may condense into homogeneous masses and nearly hexagonal (0r related) crystals with a diameter up to 14 micron and a yellow-brownish colour, giving a strongly positive PERL's reaction. In contrast to the preceding stages intranuclear crystals of this kind have been observed in one case only. After destruction of the nuclear envelope and the marginated chromatin the crystals are lying free in the cytoplasm and later on, the cytoplasm being destroyed too, they may be ingested by von Kupffer cells. All the iron containing crystals, to be found in the cytoplasm, derive from former intranuclear inclusions. The intranuclear deposits of iron containing protein are interpreted as ferritin-aggregates. It is supposed that ferritin molecules, built up in the cytoplasm, do enter the nucleus via the pores of the nuclear envelope. Such an event not only signalizes a cytopathologic reaction but in turn may give rise to such additional cytopathologic lesions as cell shrinking and cell death.     

Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli: structure in projection at 40 A resolution.

SecA is a single-chain, membrane-associated polypeptide (102 kDa) which functions as an essential component of the protein export machinery of Escherichia coli. SecA has been crystallized from ammonium sulfate as small, three-dimensional bipyramidal crystals (0.1 x 0.1 x 0.05 mm). These crystals did not demonstrate detectable diffraction of X-rays from rotating anode sources. For study by electron microscopy, individual crystals were cross-linked in glutaraldehyde and OsO4 solutions, dehydrated, embedded in epoxy resin, and sectioned normal to crystallographic axial directions inferred from the external morphology of the crystals. Fourier transformation of processed images of untilted thin sections stained with uranyl acetate and lead citrate show reflections extending to 31 A resolution. Diffraction data and reconstructed images of the projected density of the unit cell contents indicate that the bipyramidal SecA crystals belong to orthorhombic space group C222(1) with unit cell dimensions a = 414 A, b = 381 A, and c = 243 A. Filtered images and density maps of mutually orthogonal projections of the unit cell contents are consistent with a three-dimensional model in which the asymmetric unit contains eight SecA monomers. The large unit cell dimensions and packing of protein monomers suggest that SecA is crystallizing as an oligomer of either dimers or tetramers.     

Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.     

An inherited anomaly of human eosinophils and basophils.

Inclusions staining gray or blue-gray in Romanowsky treated films were found in eosinophils and basophils in a family as an autosomal dominant anomaly without overt clinical effects. Associated with the inclusions were distinctive crystals which were different from the crystalloids of eosinophil granules and from Charcot-Leyden crystals. The restriction of the anomaly to eosinophils and basophils indicates that these are related more closely to each other than to neutrophils and mast cells, and suggests the existence of a separate stem cell for eosinophils and basophils.     

Functional significance of the crystal cells in the larva of Drosophila melanogaster

Cytoplasmic crystalline inclusions are found in some larval haemocytes of Drosophila melanogaster. Blackening can be experimentally induced in these cells, and previously it was suggested that either the substrate or enzyme for the tyrosine-tyrosinase system leading to melanin production in Drosophila larvae is found in these inclusions in the crystal cells. The present report is an attempt to further localize the enzyme and substrate. Larvae have been fed on food containing alpha-C(14)-tyrosine and autoradiographs of the blood cells taken from these larvae subsequently prepared. The C(14) activity in the crystal cells is restricted to the crystal inclusions in the cells and is significantly higher than that found in the other type of haemocytes, the plasmatocytes. When samples of the blood cells are incubated in DOPA solution, the extra-crystalline cytoplasm becomes blackened while the crystals themselves remain colorless. These observations are consistent with the hypothesis that the substrate is localized in the crystal inclusions whereas enzyme is found in the surrounding cytoplasm. An in vivo structural isolation would serve to separate enzyme and substrate rather than an inhibition by dehydrogenases as postulated by previous authors. In vitro examination with the phase microscope has shown that the crystal cells rupture easily and the crystals dissolve in the haemolymph. Therefore any treatment which tends to disrupt the structural integrity of the cell will allow the enzyme and substrate to come together. Humoral factors preceding metamorphosis might account for the in vivo release of the enzymatic reaction by initially altering the permeability of the cell.     

The phase behavior of cholesteryl esters in intracellular inclusions.

Differential scanning calorimetry and polarizing light microscopy have been used to investigate kinetic and thermodynamic properties of the phase behavior of cholesteryl ester contained in Fu5AH rat hepatoma cells and J774 murine macrophages. These cultured cells store cholesteryl esters as cytoplasmic inclusions of approximately 1-micron diameter and thus are models of the foam cells characteristic of atherosclerotic plaque. Simple binary mixtures of cholesteryl palmitate and cholesteryl oleate, the predominant cholesteryl esters in cellular inclusions in both cell types serve as models to explain important aspects of the phase behavior of these inclusions. Although inclusions should exist as stable crystals at 37 degrees C under conditions of thermodynamic equilibrium, microscopic examination of cells indicates that inclusions exist as metastable liquid crystals at 37 degrees C for extended periods of time. Using an analytical model based on nucleation theory, we predict that the cholesteryl ester inclusions should be liquid-crystalline in the cytoplasm of living cells. This may not be true either for lysosomal cholesteryl ester or for extracellular cholesteryl ester present in advanced atherosclerotic plaque where fusion of droplets can enhance the possibility of crystallization. The enhanced metastability of the relatively fluid liquid-crystalline state in cellular inclusions should result in increased activity of the neutral cholesteryl ester hydrolase in living cells.