Involvement of a Cell Envelope Component of Escherichia coli in the Early Stages of Infection with Bacteriophage ϕX174

Envelope fraction I prepared from a ?X174 sensitive host, KD4301, showed a strong eclipsing activity, while the lipopolysaccharide (LPS) fraction showed a weak activity. The eclipsing activity in envelope fraction I was sensitive to heat treatment, while that in the LPS fraction was insensitive. When the complete phage particles (114S) were treated with envelope fraction I, the eclipsed particles (70S) and a rapidly sedimenting component were obtained, but when they were treated with LPS, only 70S eclipsed particles were obtained. Electron microscopic observation showed that there were two types of eclipsed particles formed on treatment with fraction I; in one of them phage DNA was extruded from the phage particles as a thick bundle, and in the other more than 95% of the phage DNA was extruded from the phage particles. The rapidly sedimenting component was the membrane-eclipsed particle complex. LPS gave only one type of eclipsed particles in which DNA was extruded as a thick bundle. These results indicate that a heat labile component in the cell envelopes other than LPS is involved in the extrusion of ?X174 DNA.     

Isolation and characterization of a new class of cytoplasmic RNP-particles from rabbit reticulocytes containing 7SL RNA

Ribonucleoprotein particles with sedimentation coefficient of 12-14S were isolated from ribosome-free extracts of rabbit reticulocytes. The particles contain one RNA molecule, whose relative electrophoretic mobility and the 3'-terminal nucleotide sequence correspond to those of the 7SL RNA from mammalian cells and one type of polypeptide chains with a molecular weight of 80,000-85,000 Da. The nucleic component of these particles is identical to that of SRP from dog pancreatic cells but differs from the latter by the protein component.     

Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.     

Reevaluation of the proteins in rabies virus particles.

Protein content and localization of individual proteins of rabies virus have been studied. Four major proteins (estimated molecular weights, about 65,000, 54,000, 37,000 and 21,000), one minor component (molecular weight, about 200,000), and one intermediate (as regards its molar concentration) component (molecular weight, about 43,000) were revealed in rabies virus particles. In subviral particles accumulating in virus-infected cells, the 200,000-, 54,000-, and 37,000-dalton components were revealed. Some properties of the subviral particles allow them to be considered as viral nucleocapsids and the proteins composing them as analogs of L, N, and NS proteins of other rhabdoviruses. Thus, the protein composition of the rabies virus strain studied does not differ from that of other rhabdoviruses.     

Ribocharin: a nuclear Mr 40,000 protein specific to precursor particles of the large ribosomal subunit

Using a monoclonal antibody (No-194) we have identified, in Xenopus laevis and other amphibia, an acidic protein of Mr 40,000 (ribocharin) which is specifically associated with the granular component of the nucleolus and nucleoplasmic 65S particles. These particles contain the nuclear 28S rRNA and apparently represent the precursor to the large ribosomal subunit in nucleocytoplasmic transit. By immunoelectron microscopy ribocharin has been localized in the granular component of the nucleolus and in interchromatin granules. During mitosis ribocharin-containing particles are associated with surfaces of chromosomes and are recollected in the reconstituting nucleoli in late telophase. We suggest that ribocharin is a specific component of precursor particles of the large ribosomal subunit, which dissociates from the 65S particle before passage through the nuclear envelope, and is reutilized in ribosome biogenesis.     

Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl.

Although hepatitis A virus (HAV) released by infected BS-C-1 cells banded predominantly at 1.325 g/cm3 (major component) in CsCl, smaller proportions of infectious virions banded at 1.42 g/cm3 (dense HAV particles) and at 1.27 g/cm3 (previously unrecognized light HAV particles). cDNA-RNA hybridization confirmed the banding of viral RNA at each density, and immune electron microscopy demonstrated apparently complete viral particles in each peak fraction. The ratio of the infectivity (radioimmunofocus assay) titer to the antigen (radioimmunoassay) titer of the major component was approximately 15-fold greater than that of dense HAV particles and 4-fold that of light HAV particles. After extraction with chloroform, the buoyant density of light and major component HAV particles remained unchanged, indicating that the lower density of the light particles was not due to association with lipids. Light particles also banded at a lower density (1.21 g/cm3) in metrizamide than did the major component (1.31 g/cm3). Dense HAV particles, detected by subsequent centrifugation in CsCl, were indistinguishable from the major component when first banded in metrizamide (1.31 g/cm3). However, dense HAV particles recovered from CsCl subsequently banded at 1.37 g/cm3 in metrizamide. Electrophoresis of virion RNA under denaturing conditions demonstrated that dense, major-component, and light HAV particles all contained RNA of similar length. Thus, infectious HAV particles released by BS-C-1 cells in vitro consist of three distinct types which band at substantially different densities in CsC1, suggesting different capsid structures with varied permeability to cesium or different degrees of hydration.     

Chicken MAR binding protein p120 is identical to human heterogeneous nuclear ribonucleoprotein (hnRNP) U.

We have previously identified two proteins from chicken oviduct nuclei that specifically bind to matrix/scaffold attachment regions (MARs/SARs). Here one of these proteins, named p120 due to its apparent molecular weight, is purified to near homogeneity and shown to be identical to a previously described component of heterogeneous nuclear ribonucleoprotein particles, hnRNP U, on the basis of amino acid sequence analysis of tryptic peptides. p120 binds to multiple MAR fragments provided they have a minimal length of approximately 700 bp. Binding of MAR fragments is specifically competed by homoribopolymers poly(G) and poly(I), which form four-stranded structures. Our results suggest that p120/hnRNP U may serve a dual function, first as a component of hnRNP particles, and second as an element in the higher-order organization of chromatin.     

Critical Arginine Residue for Maintaining the Bacteriophage Tail Structure

The addition of 0.2 m l-arginine to various T-even bacteriophage preparations inactivated the virus preparations irreversibly. The virus particles were even more sensitive to added d-arginine and l-homoarginine than to l-arginine but were unaffected by arginine analogues with either an altered carboxyl group or guanidyl group. Treatment of phage T2H with 2,3-butanedione, a reagent which specifically reacts with the guanidyl portion of arginine residues, resulted in the apparent in-activation of most of the virus particles. However, after incubation of the treated particles at pH 7.5 at 37 C for 1 hr in the absence of butanedione, the original virus titer almost completely returned. The reactivation was completely inhibited by the presence of 0.2 m d-arginine. It appeared that the virus protein coat was sufficiently plastic so that the initial conformational change resulting from the alteration of an arginine residue (to possibly an ornithine residue) was at least partially reversible and that the virus tail proteins then refolded to produce a stable and active virus particle. These reactivated virus particles were not sensitive to inactivation by d-arginine but could now be rapidly inactivated by l-ornithine. Virus particles inactivated by arginine have altered tail structures. They have contracted tail sheaths still attached to tail plates and still contain tail cores. These properties of virus particles indicate that there is a free carboxyl group and a guanidyl group spatially equivalent to an arginine residue on one component of the virus tail which bind reversibly by means of polar linkages to another tail component. These bonds maintain the integrity of the virus tail. Added arginine appears to compete with this endogenous viral arginine for the binding sites and then to favor an irreversible conformational change.     

Receptor-mediated mechanisms of lipoprotein remnant catabolism

Chylomicron and VLDL are triglyceride-rich lipoprotein particles assembled by the intestine and liver respectively. These particles are not metabolized by the liver in their native form. However, upon entry into the plasma, their triglyceride component is rapidly hydrolyzed by lipoprotein lipase and they are converted to cholesterol-rich remnant particles. The remnant particles are recognized by the liver and rapidly cleared from the plasma. This process is believed to occur in two steps. (i) An initial sequestration of remnant particles on hepatic cell surface proteoglycans, and (ii) receptor-mediated endocytosis of remnants by hepatic parenchymal cells. The initial binding to proteoglycans may be facilitated by lipoprotein lipase and hepatic lipase which possess both lipid- and heparin-binding domains. The subsequent endocytic process may be mediated by LDL receptors and/or LRP. Both receptors have a high affinity for apoE, a major apolipoprotein component of remnant particles. The lipases may also serve as ligands for these receptors. An impairment of any component of this complex process may result in an accumulation of remnant particles in the plasma leading to atherosclerosis and coronary heart disease.     

Bacteriophage T4 baseplate components. II. Binding and location of bacteriophage-induced dihydrofolate reductase.

The location of T4D phage-induced dihydrofolate reductase (dfr) has been determined in intact and incomplete phage particles. It has been found that phage mutants inducing a temperature-sensitive dfr (dfrts) procude heat-labile phage particles. The structural dfr produced by these ts mutants was shown to assume different configurations depending on the temperature at which the phage is assembled. Morphogenesis of incomplete phage particles lacking the gene 11 protein on their baseplates was found to be inhibited by reagents binding to dfr, such as antibodies to dfr. Further, cofactor molecules for dfr, such as reduced nicotinamide adenine dinucleotide phosphate and reduced nicotinamide adenine dinucleotide, also inhibited the step in morphogenesis involving the addition of gene 11 product. On the other hand, inhibitors of dfr, such as adenosine dephosphoribose, stimulated the addition of the gene 11 protein. It has been concluded that the phage-induced dfr is a baseplate component which is partially covered by the gene 11 protein. The properties of phage particles produced after infection of the nonpermissive host with the one known T4D mutant containing a nonsense mutation in its dfr gene suggested that these progeny particles contained a partial polypeptide, which was large enough to serve as a structural element.     

Effect of ATP on the EPR spectrum at 20°K of phosphorylating sub-mitochondrial particles

By measurements of the EPR spectrum of substrate-reduced anaerobic phosphorylating sub-mitochondrial particles at 20°K, ATP was found to bring about the oxidation of four components (two iron-sulphur proteins associated with NADH dehydrogenase, and two unidentified iron proteins) and the reduction of one component. Thus energization of the particles lowers the effective redox potential of four components and raises that of a fifth.     

RNA associated with murine intracisternal type A particles codes for the main particle protein.

Intracisternal type A particles were isolated from MOPC-104E myeloma grown subcutaneously and from N 4 neuroblastoma cells in culture. Polyadenylated RNA was prepared from the particles and tested in a cell-free translation system derived from rabbit reticulocytes. RNA from the two sources directed the synthesis of multiple polypeptides with similar distributions of electrophoretic mobilities in sodium dodecyl sulfate-containing polyacrylamide gels, including one conponent of the same size as the major A-particle structural protein (73,000 daltons). Analysis of the RNAs by electrophoresis in methyl mercury-containing agarose gels revealed a 35S component common to A-particles from both cell types. This was a major component of the N4 preparations, whereas a 28S species predominated in the case of MOPC-104E. These two RNAs (35S from N4 cells and 28S from MOPC-104E), when isolated on isokinetic sucrose gradients, each directed the synthesis of a 73,000-molecular-weight polypeptide that comigrated on gels with authentic A-particle structural protein. Idnetity of the cell-free product was confirmed by two-dimensional analysis of the [35S]methionine-labeled tryptic peptides. The N4 RNA preparations also contained a major32S component which did not code effectively for the A-particle structural protein.     

Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.     

Effect of different neutral phospholipids on apolipoprotein binding by artificial lipid particles in vivo

Soybean triacylglycerol particles, stabilized with egg yolk sphingomyelin (SPH), phosphatidylcholine (PC), phosphatidylethanolamine (PE), or PC-PE mixtures, with diameters ranging from 170 to 550 nm were prepared by sonication and isolated by ultracentrifugation. Binding of apoproteins to the lipid particles was studied in vivo using the strategy of Connelly and Kuksis. The recoveries of the injected particles, which had decreased in size and undergone minimal changes in lipid composition, ranged from 70% and 57% for SPH- and PC-stabilized particles to 14% for particles stabilized with egg yolk PC-PE mixtures. The apoprotein (apo) composition of the recovered particles showed qualitative and quantitative differences, which were affected by the number of washes during isolation. After four washes, the SPH and PC particles contained apoE, apoC-II, and apoC-III as major components and apoA-IV as minor components. In addition, all particles, except those stabilized with egg yolk PC, contained large amounts of albumin. In contrast to egg yolk PC, the dipalmitoyl PC particles bound albumin as a major component. The recovered PC-PE and PE particles were characterized by a relative decrease of apoC and greatly increased binding of albumin. The higher rate of clearance of the PE-containing particles was attributed to a relative absence of apoC-III, which is known to delay hepatic uptake of lipid particles containing it, and to a more rapid hydrolysis of PE by lipoprotein lipases. Since PE occurs as a minor and variable component of chylomicrons and plasma lipoproteins, the present observations are of physiological interest.     

Comparison of In Vitro and Cell-Mediated Alteration of a Human Rhinovirus and Its Inhibition by Sodium Dodecyl Sulfate

After human rhinovirus type 2 (HRV-2) attaches to HeLa cells, two types of subviral particles are formed which closely resemble particles produced in vitro by acid or heat. One type of particle contains RNA whereas the second sediments as an empty capsid and is RNA-deficient. Sodium dodecyl sulfate (SDS) at 10(-4) M inhibits the cell-mediated formation of these particles from HRV-2 virions and the ability of HRV-2 to form plaques, but it does not inhibit the formation of plaques by human rhinovirus 14 (HRV-14). SDS also stabilizes HRV-2 against inactivation by acid or heat to a much greater extent than it does HRV-14. In a similar manner, SDS protects against the acid inactivation of the subpopulation of HRV-2 natural top component particles which attach to virus-specific cellular receptors. This suggests that the loss of native properties of natural top component particles and of virion are related processes. The basis for this alteration and also its role in infection are discussed.     

Biochemical research on oogenesis. Comparison between the proteins of the ribosomes and the proteins of the 42 S particles from small oocytes of Xenopus laevis

Previtellogenic oocytes of Xenopus laevis synthesize large amounts of 5 S RNA and transfer RNA, but very little, if any, 28 S and 18 S RNA. About half of the RNA of these oocytes is stored in nucleoprotein particles sedimenting at 42 S. These particles contain 5 S RNA, transfer RNA, and several proteins, the function of which remains so far unknown.The proteins of the 42 S particles were analyzed by two-dimensional electrophoresis on polyacrylamide gel. The resulting fingerprints displayed one major and two minor basic spots. None of these coincided with any of the 37 spots produced by the 60 S subunit of the ribosomes and with the 30 spots produced by the 40 S subunit. We conclude that no ribosomal component other than 5 S RNA is present in the 42 S particles.The fingerprints of 40 S and 60 S ribosomal proteins from X. laevis coincided almost completely with the corresponding fingerprints from the rat and the rabbit.     

Glycophorin-enriched vesicles obtained by a selective extraction of human erythrocyte membranes with a non-ionic detergent.

A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles.     

Study on the Energy Transfer Process Between the Photosystem Ⅱ Reaction Center and Light-Harvesting System

The primary reaction kinetics of the photosystem Ⅱ particles isolated from spinach (Spinacia oleracea Mill. ) have been studied using the subpicosecond transient absorption technique. Three lifetime components, (0.76±0.50) ps, (8.70±2.00) ps and (138.00±20.00) ps, were obtained by the multi-exponential curval fitting. When the samples were exposed to strong light for one hour, only one component, 133 ps, was found. It was proposed that the 760 fs component was most probably attributed to the energy transfer from light-harvesting system to the reaction center.     

Endocytosis by platelets during cationized ferritin-induced aggregation

Summary Localization of cationized ferritin (CF) particles in the process of CF-induced aggregation of rabbit platelets was investigated by electron microscopy. CF particles attached to the surface membrane of discoidal platelets immediately after the addition of CF. Some platelets were connected to each other through the CF particles located on their surfaces. At 30 s after the addition of CF, aggregation of platelets in round form was observed. During the time course of aggregation, CF particles moved to the interplatelet spaces. Also CF particles were found in the open canalicular system, the membrane component of which was stained with ruthenium red. On the other hand, CF particles were also found in ruthenium-red-negative vesicles in platelets. At 180 s after, CF particles containing vacuoles, which showed acid phosphatase activity, were observed in the aggregates. These results suggest that part of CF particles may be incorporated into the cytoplasma by endocytosis.     

Luminescent magnetic particles: structures, syntheses, multimodal imaging, and analytical applications

Luminescent magnetic particles (LuMaPs) are attractive tools for life science applications such as multimodal imaging, analyte monitoring, nanotherapeutics, and combinations thereof. LuMaPs consist of at least one magnetic and one luminescent component which often are incorporated in a (polymeric) matrix. Alarge variety of materials do exist for the components that make up LuMaPs. However, a smart selection and combination is required for achieving useful tools. While the magnetic component mainly influences the response to a magnetic field, the luminophore can act as label, sensor, or therapeutic agent. The matrix fulfills tasks such as stabilizing the luminophore and magnet, carrying useful functional groups on its surface, or hosting smart drug delivery systems. Surface modifications with targeting ligands can further improve the applicability of LuMaPs, for example in biomedicine. This review provides an overview on LuMaPs with respect to the materials used and to its structures. Routes toward LuMaPs are outlined, and potential applications are discussed.