Molecular simulation study of assembly of DNA-grafted nanoparticles: effect of bidispersity in DNA strand length

In this paper, we use molecular dynamics simulations to study the assembly of DNA-grafted nanoparticles to demonstrate specifically the effect of bidispersity in grafted DNA strand length on the thermodynamics and structure of nanoparticle assembly at varying number of grafted single-stranded DNA (ssDNA) strands and number of guanine/cytosine (G/C) bases per strand. At constant number of grafted ssDNA strands and G/C nucleotides per strand, as bidispersity in strand lengths increases, the number of nanoparticles that assemble as well as the number of neighbours per particle in the assembled cluster increases. When the number of G/C nucleotides per strand in short and long strands is equal, the long strands hybridise with the other long strands with higher frequency than the short strands hybridise with short/long strands. This dominance of the long strands leads to bidisperse systems having similar thermodynamics to that in corresponding systems with monodisperse long strands. Structurally, however, as a result of long–long, long–short and short–short strand hybridisation, bidispersity in DNA strand length leads to a broader inter-particle distance distribution within the assembled cluster than seen in systems with monodisperse short or monodisperse long strands. The effect of increasing the number of G/C bases per strand or increasing the number of grafted DNA strands on the thermodynamics of assembly is similar for bidisperse and monodisperse systems. The effect of increasing the number of grafted ssDNA strands on the structure of the assembled cluster is dependent on the extent of strand bidispersity because the presence of significantly shorter ssDNA strands among long ssDNA strands reduces the crowding among the strands at high grafting density. This relief in crowding leads to larger number of strands hybridised and as a result larger coordination number in the assembled cluster in systems with high bidispersity in strands than in corresponding monodisperse or low bidispersity systems.     

On the Determination of the Pattern of Vascular Tissue in Peas

This work is concerned with the rules determining the placeof joining of two vascular strands. Auxin can induce the differentiationof vascular tissue, and this fact is used here for an experimentalstudy of the spatial interactions of vascular strands. Differentiated vascular tissue whose source of auxin has beenremoved attracts newly induced vascular strands. This attractionis expressed in the joining of the new strands to the pre-existingvascular tissue. Differentiated vascular tissue which is wellsupplied with auxin inhibits rather than attracts the formationof new vascular strands in its vicinity. Experiments on pea apices have extended these results to naturallyinduced vascular strands. It is shown that when a leaf primordiumis damaged at an early age its vascular strands are joined bythe strands induced by new leaves, and the contacts may be formedacross the leaf gap. The joining of the vascular strands is,therefore, much closer to the leaf than is normal and this isprobably due to the reduction in the supply of auxin from thedamaged leaf to its vascular traces. It is also shown that whena lateral bud grows its vascular traces join preferentiallythe vascular system leading to organs which have been removed.These vascular traces of a bud specifically avoid the vascularsystem of a leaf or a shoot which is still growing and producingauxin. These results are discussed in reference to the relation betweenthe vascular system and phyllotaxis and to the existence ofleaf gaps.     

Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells.

We have developed a ribonuclease protection method suitable for sensitive detection of an RNA species in the presence of a large excess of its complementary strand, as for the detection of negative strands of positive-strand RNA viruses. By using this method to probe for poliovirus negative strands in virions, we found that positive strands are present in at least 40,000-fold excess over negative strands. Thus, we have confirmed that poliovirus encapsidation is highly specific for positive strands and have demonstrated that the genome-linked protein VPg, which is covalently attached to the 5' ends of both positive and negative strands, cannot be the sole determinant of RNA packaging. We tested the ratios of viral positive strands to negative strands in cells at different times during infection; this value ranged from approximately 40/1 to 70/1, being highest at 4 h and lower at 2 and 6 h postinfection.     

Transcellular Strands in Sieve Tubes; What Are They?

We show that sieve elements of Nymphoides peltata (S. G. Gmel.)O. Kuntze contain strands which are bundles of P-protein filaments.We observe the strands under the light microscope (differential-interferencecontrast), and in the scanning electron microscope which showssome of them to be arranged as a parietal network. We find bundlesof filaments which correspond to these strands in sections ofembedded sieve elements in the transmission electron microscope,and also in freeze-fracture replicas of sieve elements in vascularbundles frozen intact while translocating carbon-14. Not allthe strands are necessarily transcellular; some may end in theparietal layer just to the inside of the plasmalemma where theyappear to come in contact with membranes, possibly of endoplasmicreticulum. The filaments in the strands have the same bandedappearance as filaments in the sieve pores. We are unable tofind any membrane or other special boundary round the strands;we propose they should be called ‘filamentous strands’.We suggest that the filaments are aggregated into strands bythe Bernoulli effect when fluid flows through sieve elements.We suggest that the strands may be formed by flow during translocationas well as by flow due to injury.     

Studies on biosynthesis of tobacco mosaic virus. VII. Radioactivity of plus and minus strands in different forms of viral RNA after labelling of infected tobacco leaves

Tobacco leaves were labelled with tritiated undine for 30 or 120 minutes at different times after systemic infection with tobacco mosaic virus. RNA was extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus strands (complementary RNA) was determined in each fraction by an isotope dilution assay. The amount of minus strands in the RP and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined.Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms as during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small.The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated.In RF and RI the radioactivity in plus strands was between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral RNA.     

Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis

Members of the newly identified claudin gene family constitute tight junction (TJ) strands, which play a pivotal role in compartmentalization in multicellular organisms. We identified oligodendrocyte-specific protein (OSP) as claudin-11, a new claudin family member, due to its sequence similarity to claudins as well as its ability to form TJ strands in transfected fibroblasts. Claudin-11/OSP mRNA was expressed in the brain and testis. Immunofluorescence microscopy with anti-claudin-11/OSP polyclonal antibody (pAb) and anti-neurofilament mAb revealed that in the brain claudin-11/OSP-positive linear structures run in a gentle spiral around neurofilament-positive axons. At the electron microscopic level, these linear structures were identified as the so-called interlamellar strands in myelin sheaths of oligodendrocytes. In testis, well-developed TJ strands of Sertoli cells were specifically labeled with anti-claudin-11/OSP pAb both at immunofluorescence and electron microscopic levels. These findings indicated that the interlamellar strands of oligodendrocyte myelin sheaths can be regarded as a variant of TJ strands found in many other epithelial cells, and that these strands share a specific claudin species, claudin-11/OSP, with those in Sertoli cells to create and maintain the repeated compartments around axons by oligodendrocytes.     

Heterogeneity of tight junctions along the collecting duct in the renal medulla

Summary The tight junctions along the medullary collecting duct in the kidneys of the rat and the rabbit were studied with freeze-fracture electron microscopy and quantitated according to the number of strands and the apico-basal depth (nm) of the junctions.The most elaborate tight junctions were found in the inner stripe of the outer medulla; rat: 10.6±0.8 strands and 205±24nm; rabbit: 11.6±2.4 strands and 291±55 nm.The elaboration of the tight junctions decreased continuously towards the papillary tip. Inner zone I; rat: 9.3±2.6 strands and 186±38nm, rabbit: 9.5±2.3 strands and 247±59nm. Inner zone II; rat: 7.1±2.2 strands and 129±32nm, rabbit: 8.5±1.4 strands and 199±26nm. Inner zone III; rat: 6.0±1.6 strands and 111 + 19 nm, rabbit: 7.0±1.5 strands and 183±43 nm. In the inner zone III comprising the papillary tip tight junctions with only 1–3 strands were not infrequently seen. Preliminary findings in the kidney of the golden hamster indicate a similar decline of junctional tightness along the collecting duct.These morphological observations suggest that the permeability of the paracellular pathway of the medullary collecting duct increases towards the tip of the papilla, especially in the rat. The functional implications for the medullary recycling of urea and electrolytes, and for the urinary concentrating mechanism are discussed.In addition, the tight junctions of the papillary epithelium are described.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

Beta edge strands in protein structure prediction and aggregation

It is well established that recognition between exposed edges of beta-sheets is an important mode of protein-protein interaction and can have pathological consequences; for instance, it has been linked to the aggregation of proteins into a fibrillar structure, which is associated with a number of predominantly neurodegenerative disorders. A number of protective mechanisms have evolved in the edge strands of beta-sheets, preventing the aggregation and insolubility of most natural beta-sheet proteins. Such mechanisms are unfavorable in the interior of a beta-sheet. The problem of distinguishing edge strands from central strands based on sequence information alone is important in predicting residues and mutations likely to be involved in aggregation, and is also a first step in predicting folding topology. Here we report support vector machine (SVM) and decision tree methods developed to classify edge strands from central strands in a representative set of protein domains. Interestingly, rules generated by the decision tree method are in close agreement with our knowledge of protein structure and are potentially useful in a number of different biological applications. When trained on strands from proteins of known structure, using structure-based (Dictionary of Secondary Structure in Proteins) strand assignments, both methods achieved mean cross-validated, prediction accuracies of approximately 78%. These accuracies were reduced when strand assignments from secondary structure prediction were used. Further investigation of this effect revealed that it could be explained by a significant reduction in the accuracy of standard secondary structure prediction methods for edge strands, in comparison with central strands.     

Structural variation in the glycan strands of bacterial peptidoglycan

The normal, unmodified glycan strands of bacterial peptidoglycan consist of alternating residues of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. In many species the glycan strands become modified after their insertion into the cell wall. This review describes the structure of secondary modifications and of attachment sites of surface polymers in the glycan strands of peptidoglycan. It also provides an overview of the occurrence of these modifications in various bacterial species. Recently, enzymes responsible for the N-deacetylation, N-glycolylation and O-acetylation of the glycan strands were identified. The presence of these modifications affects the hydrolysis of peptidoglycan and its enlargement during cell growth. Glycan strands are frequently deacetylated and/or O-acetylated in pathogenic species. These alterations affect the recognition of bacteria by host factors, and contribute to the resistance of bacteria to host defence factors such as lysozyme.     

Changes in Hechtian strands in cold-hardened cells measured by optical microsurgery

Optical microsurgical techniques were employed to investigate the mechanical properties of Hechtian strands in tobacco (Nicotiana tabacum) and Ginkgo biloba callus cells. Using optical tweezers, a 1. 5-microm diameter microsphere coated with concanavalin A was inserted though an ablated hole in the cell wall of a plasmolyzed cell and attached to a Hechtian strand. By displacing the adhered microsphere from equilibrium using the optical trapping force, the tensions of individual strands were determined. Measurements were made using both normal and cold-hardened cells, and in both cases, tensions were on the order of 10(-12) N. Significant differences were found in the binding strengths of cold-hardened and normal cultured cells. An increased number density of strands in cold-hardened G. biloba compared with normal cultured cells was also observed. Although no Hechtian strands were detected in any Arabidopsis callus cells, strands were present in leaf epidermal cells. Finally, the movement of attached microspheres was monitored along the outside of a strand while cycling the osmotic pressure.     

Occludin and claudins in tight-junction strands: leading or supporting players?

Tight junctions have attracted much interest from cell biologists, especially electron microscopists, since on freeze-fracture electron microscopy they appear as a well-developed network of continuous, anastomosing intramembranous strands (tight-junction strands). These strands might be directly involved in the 'barrier' as well as 'fence' functions in epithelial and endothelial cell sheets, but until recently little was known of their constituents. This review discusses current understanding of the molecular architecture of tight-junction strands, focusing on the recent discovery of two distinct types of tight-junction-specific integral membrane proteins, occludin and claudins.     

Manner of interaction of heterogeneous claudin species within and between tight junction strands

In tight junctions (TJs), TJ strands are associated laterally with those of adjacent cells to form paired strands to eliminate the extracellular space. Claudin-1 and -2, integral membrane proteins of TJs, reconstitute paired TJ strands when transfected into L fibroblasts. Claudins comprise a multigene family and more than two distinct claudins are coexpressed in single cells, raising the questions of whether heterogeneous claudins form heteromeric TJ strands and whether claudins interact between each of the paired strands in a heterophilic manner. To answer these questions, we cotransfected two of claudin-1, -2, and -3 into L cells, and detected their coconcentration at cell-cell borders as elaborate networks. Immunoreplica EM confirmed that distinct claudins were coincorporated into individual TJ strands. Next, two L transfectants singly expressing claudin-1, -2, or -3 were cocultured and we found that claudin-3 strands laterally associated with claudin-1 and -2 strands to form paired strands, whereas claudin-1 strands did not interact with claudin-2 strands. We concluded that distinct species of claudins can interact within and between TJ strands, except in some combinations. This mode of assembly of claudins could increase the diversity of the structure and functions of TJ strands.     

The relationship between microtubules and chromatin in spermatids ofAcrididae

Summary A frequent coincidence of perinuclear microtubules and chromatin strands at nuclear membrane level was observed in spermatids ofChorthippus apicalis andOedipoda coerulescens. More developed spermatids also show apparent continuity between these chromatin strands and microtubules. We would suggest the importance of this relationship in the arrangement adopted by these strands.     

Development of tight junctions in the caput epididymal epithelium of the mouse

The course of development of the epithelial tight junctions of the Wolffian duct and the caput epididymal principal cells in the mouse were examined by freeze-fracture. The histogenesis of the epididymis is briefly described. In the 12-day embryo, tight junction meshworks surround the entire circumference of the columnar cells in the juxtaluminal position. During fetal life, the strands are more discontinuous than those of postnatal mice, and two or more strands frequently run together. Up to 10 days of age, the basal compartments of the tight junctions are much larger than the luminal ones. Marked increases in both the number of strands and the depth of the tight junctions appear by 20 days. Strands with a terminal loop are often observed up to 16 days, except for the newborn stage, suggesting that the formation of the terminal loop is related to the active elongation of the strands. The tight junctions increase greatly in number and depth near three-cell junctions. Up to 20 days, the strands anastomose frequently, with no particular orientation to the cell axis. After 20 to 37 days, the direction of the strands becomes parallel to the luminal surface, with a decreased number of anastomoses as the lumen widens. In the adult, the number of sealing strands is about 10 within the depth of the tight junctions. Free-ended strands are seen in all stages examined. The formation of the tight junction meshworks is discussed in the light of the findings during the development.     

Chromosomal sites of DNA-membrane attachment in Escherichia coli

Evidence is presented to show that both the chromosomal replication point and the chromosomal origin in Escherichia coli are associated with a structure possessing the sedimentation properties and enzyme sensitivities characteristic of membrane. The data suggest that both newly synthesized DNA strands and template DNA strands are bound at this replication point and that both strands are also bound at the origin.     

Polysaccharide/polynucleotide complexes. Part 6: complementary-strand-induced release of single-stranded DNA bound in the schizophyllan complex

Spectroscopic properties of single-stranded DNA/schizophyllan ternary complexes (ss-DNA2s-SPG), induced by addition of either complementary or noncomplementary strands, have been investigated. The addition of the complementary strands to ss-DNA2s-SPG induced the quick release of the bound ss-DNA to the complementary strands (both DNA and RNA), whereas the ternary complex was unaffected upon addition of noncomplementary strands. Our experiments imply that SPG has complexation properties indispensable to the gene carriers. As far as we know, there is no report on exploitation of such nonviral gene carriers that can accomplish an intelligent release of the bound ss-DNA toward the complementary strands. We believe, therefore, that SPG, a natural and neutral polysaccharide, has a great potential to become a new ss-DNA carrier.     

Separation of complementary strands of plasmid DNA using the biotin-avidin system and its application to heteroduplex formation and RNA/DNA hybridizations in electron microscopy.

A method for the separation of complementary strands with the help of the biotin-avidin system is described. Restriction fragments were terminally labeled at both ends with biotinylated nucleotides. The DNA was cut by a second restriction enzyme, and the fragments were bound to an avidin agarose column. The non-biotinylated strands were eluted with 0.1 M NaOH, and the biotin-labeled strands were subsequently released from the column by elution with 50% guanidine isothiocyanate/formamide. Contamination of the separated strands by complementary single strands was less than 4%.-Separated linear single strands of the vector pEMBL were prepared. On annealing with recombinant circular DNA a substitution loop is formed which provides position and orientation markers for the unambiguous electron microscopic analysis of heteroduplexes or hybrids formed with the inserted sequences. -The terminal biotin label was visualized by complex formation with a streptavidin-ferritin conjugate.     

Leaf vasculature in Zea mays L.

The vascular system of the Zea mays L. leaf consists of longitudinal strands interconnected by transverse bundles. In any given transverse section the longitudinal strands may be divided into three types of bundle according to size and structure: small, intermediate, large. Virtually all of the longitudinal strands intergrade structurally however, from one bundle type to another as they descend the leaf. For example, all of the strands having large-bundle anatomy appear distally as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. Only the large bundles and the intermediates that arise midway between them extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of longitudinal bundles at the base of the blade, both the total and mean cross-sectional areas of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.