Large Tubular Structures Research Articles

Self-assembly of DNA parallel double-crossover motifs.

DNA double-crossover motifs, including parallel and antiparallel crossovers, serve as the structural foundation for the creation of diverse nanostructures and dynamic devices in DNA nanotechnology. Parallel crossover motifs have unique advantages over the widely used antiparallel crossover design but have not developed as substantially due to the difficulties in assembly. Here we created 29 designs of parallel double-crossover motifs varying in hybridization pathways, central domain lengths, and crossover locations to investigate their assembly mechanism. Arrays were successfully formed in four distinct designs, and large tubular structures were obtained in seven designs with predefined pathways and central domains appoximately 16 nucleotides in length. The nanotubes obtained from parallel crossover design showed improved nuclease resistance than the ones from the antiparallel counterpart design. Overall, our study provides a basis for the development of generalized assembly rules of DNA parallel crossover systems and opens new opportunities for their potential use in biological systems.

“Complex” hepatic echinococcosis

Aim. To study the long-term results of surgical treatment in patients with “complex” hepatic echinococcosis.Materials and methods. The results of surgical treatment of 118 patients with hepatic echinococcosis from 2015 to 2020 at the A.V. Vishnevsky National Medical Research Center of Surgery were analyzed. The term “complex” hepatic echinococcosis has been proposed. A comparative analysis of the number and type of complications and surgical interferences 2 groups of patients was carried out: 66 patients with hepatic echinococcosis, 52 patients with complex hepatic echinococcosis.Results. During the observation period, no any recurrence was recorded. 55 (44%) of 118 patients had complex hepatic echinococcosis. Isolated liver damage was found in 74% of cases, combined liver and lung damage in 20% cases and with other organs – in 6%. In most cases, pericystectomy was performed (82%), 8% of patients underwent atypical hepatectomy, 4% – segmental resection, 3% – laparoscopic intervention, 3% – hemihepatectomy. Postoperative complications were recorded in 22 (18,6%) of cases. Accumulations of bile and biliary fistulas formed in 45% of cases, pneumotothorax or hydrothorax – in 23%, wound abscess – in 18%, hematomas in the hepatectomy zone – in 14% of the cases.Conclusion. Surgical treatment of complex hepatic echinococcosis requires an individual approach to the choice of the operation option. Preference should be given to parenchyma-preserving radical operations. When the liver lobe is totally replaced with a hydatid cyst and in case of cystobiliary fistulas hemihepatectomy should be performed, and it is acceptable to leave the fibrous capsule on large tubular structures.

Dynamic Collapse of Underwater Metallic Structures – Recent Investigations: Contributions after the 2011 Murray Lecture

The Murray Lecture that was delivered in 2011 included a historical perspective on salient features of research conducted and published by Arun Shukla. A review of experimental work since the Murray Lecture is presented in this paper on the topic of imploding underwater structures. Specifically, the dynamic underwater collapse of metallic structures was studied under free-field and confined environments using novel applications of the Digital Image Correlation (DIC) technique. During these implosion experiments, the implodable volumes became unstable under hydrostatic and combined hydrostatic-explosive conditions. Moreover, there are two types of confinements explored in this paper. A semi-confinement, where a large tubular structure is open to a reservoir at one end and closed at the other end, which leads to water hammer waves on the closed end after implosions. Also, a full-confinement, where a large tubular structure is closed at both ends, which leads to limited hydrostatic potential energy during the implosion. For accurate displacement and velocity measurements of the collapsing structures, the 3D DIC technique is calibrated for underwater measurements in a small-scale setup for each experimental configuration. High-speed cameras are then used to record the dynamic implosion event while dynamic pressure transducers measure the emitted pressure pulses. The results of these experimental series show that the 3D DIC technique can be successfully used for displacement measurements of submerged objects by extracting intrinsic and extrinsic parameters using a submerged calibration grid. The implodable volume in an open-ended semi-confining structure displays a reduced collapse velocity with respect to a free-field configuration. This is primarily caused by a near-field pressure drop from the superposition of low-pressure implosion waves inside confining structure. Semi-confined implosion also exhibits high-pressure hammer pulses at the closed end. Implosions in fully confined vessels show significant reduction/delays in the implosion process due to limited energy present in the vessel. Lastly, during fully confined explosive experiments, the shock wave impact leads to structural vibrations in the implodable volume. The amplitude of these vibrations increases with higher hydrostatic pressures until the implodable volume shows an instability.

Abstract A11: Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo

Abstract Introduction: The exocrine pancreas comprises a branched network of ducts that are connected to acini and lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesenchyme. The formation and maturation of pancreatic ductal cells during branching morphogenesis is controlled by a complex hierarchy of transcription factors, which is not fully understood. The Ets-transcription factor Etv5 has been reported to play an important role during the development of organs that undergo branching morphogenesis such as mammary and salivary glands. We therefore aimed to characterize the functional role of Etv5 in pancreatic ductal development and differentiation. Methods: Cells were isolated from wild-type mouse pancreata and lentiviral transduction was used to induce Etv5 overexpression. Cells were grown in 3D organotypic culture and analyzed with time lapsed microscopy for morphology and dynamics in the formation of duct-like cystic structures. Differentiation status of cells grown in 3D was analyzed by immunofluorescence-staining (IF) and qPCR. To asses the role of Etv5 in the normal mouse pancreas and its role in inflammation and regeneration in vivo, Pdx1cre;Etv5-/-; RosaYFP mice were generated and aged up to six months and subjected to a cerulein induced acute pancreatitis protocol. Results: Normal pancreatic ductal cells grown in 3D-organotypic culture form spheroid cysts that resemble pancreatic ductal structures. Etv5 overexpression leads to early and exuberant formation of spheroid cysts within 2 days after seeding. Time-lapse microscopy demonstrated a significantly increased movement of cellular structures along the cyst as well as fusion of cysts into larger tubular structures. mRNA analysis of cysts harvested at day 7 displayed a strong upregulation of Sox9 and Foxa2, important regulators of ductal differentiation. Concurrently, E-cadherin was upregulated significantly whereas N-cadherin was downregulated indicating the terminal differentiation of these cells. IF-staining revealed co-localization of Etv5 and Sox9 in spheroid cysts of Etv5-overexpressing cells. Knockdown of Sox9 in Etv5-overexpressing cells with siRNA partially abrogated the formation of tubular structures and disrupted cyst architecture. Etv5 knockout mice (Pdx1Cre;Etv5-/-) were generated and we performed a cerulein-induced acute pancreatitis model. We found significantly elevated levels of serum amylase in Etv5-/- mice on Days 1 and 3 of the protocol. In a semiquantitative, blinded review of the histology, there was significantly more intense edema, inflammation, vacuolization and necrosis in Etv5-/- mice in all time points, which was also confirmed by quantitative amylase area scoring. Conclusion: Our data suggest a novel role for Etv5 in pancreatic ductal morphogenesis and lumen formation that is at least in part mediated by Sox9. In addition, our data suggests that the loss of Etv5 expression increases susceptibility to pancreatitis and results in persistent pancreatitis with delayed regeneration. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. Ets transcription factor Etv5 regulates ductal morphogenesis and differentiation in association with Sox9 in vitro and increases susceptibility and delays recovery from pancreatitis in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A11.

Methodology for the accuracy improvement of FEM beam type T-junctions of buses and coaches structures

The analysis of structures with finite elements methods (FEM) represents a widely spread technique. For large tubular structures similar to the buses and coaches upper structures, beam type elements are utilized due to the fact that these elements provide satisfactory results at relatively reduced computational performances. However the beam type elements have a main disadvantage determined by the fact that the modeled joints have an infinite rigid behavior. This shortcoming determines a stiffer behavior of the modeled structures which translates into an error source for the structural simulations (up to 45%). To overcome this problem, a simple methodology was conceived and an alternative optimized equivalent beam model obtained. The methodology studies the behavioral characteristics of beam modeled T-junctions determining their limitations and comparing them to equivalent T-junctions modeled with shell and volume elements. This way an improved Tjunction has been obtained, in which the behavioral error was reduced to less than 5%. Furthermore the FEM obtained results were validated with real T-junctions.

Nodulation of Aeschynomene afraspera and A. indica by Photosynthetic Bradyrhizobium Sp. Strain ORS285: The Nod-Dependent Versus the Nod-Independent Symbiotic Interaction

Here, we present a comparative analysis of the nodulation processes of Aeschynomene afraspera and A. indica that differ in their requirement for Nod factors (NF) to initiate symbiosis with photosynthetic bradyrhizobia. The infection process and nodule organogenesis was examined using the green fluorescent protein-labeled Bradyrhizobium sp. strain ORS285 able to nodulate both species. In A. indica, when the NF-independent strategy is used, bacteria penetrated the root intercellularly between axillary root hairs and invaded the subepidermal cortical cells by invagination of the host cell wall. Whereas the first infected cortical cells collapsed, the infected ones immediately beneath kept their integrity and divided repeatedly to form the nodule. In A. afraspera, when the NF-dependent strategy is used, bacteria entered the plant through epidermal fissures generated by the emergence of lateral roots and spread deeper intercellularly in the root cortex, infecting some cortical cells during their progression. Whereas the infected cells of the lower cortical layers divided rapidly to form the nodule, the infected cells of the upper layers gave rise to an outgrowth in which the bacteria remained enclosed in large tubular structures. Together, two distinct modes of infection and nodule organogenesis coexist in Aeschynomene legumes, each displaying original features.

Formation of abnormally large-sized tubular amyloid β aggregates on a nanostructured gold surface

The effect of the properties of a nanostructured gold surface (nano-Au surface) on the aggregation of Amyloid β(1–40) (Aβ40) was investigated. A nano-Au surface, in the form of immobilized nanoparticles, was prepared by using a thermal evaporator, resulting in the formation of nanosized clusters with sizes less than 10 nm. When Aβ40 was incubated with the nano-Au surface, abnormally large-sized tubular aggregates were formed on the surface and typical fibril formation was suppressed in the solution. This abnormally large tubular structure represents a novel type of Aβ40 aggregate. In the absence of the nano-Au surface, the diameters of the Aβ40 fibrils were less than 10 nm. However, the height of the tubular aggregates formed on a nano-Au surface was 80–100 nm. Such large-sized aggregates of Aβ40 have not been reported in previous studies dealing with interactions of suspended nanoparticles with proteins. This can be attributed to differences in the aggregation mechanism between immobilized and suspended nanoparticles. The formation of Aβ40 aggregates by nano-Au surface will provide the possible mechanism for abnormal fibril formation.

On the organisation of vesicular apparatus in poultry lice Lipeurus lawrensis tropicalis PETERS (Phthiraptera: Ischnocera)

The vesicular apparatus of Lipeurus lawrensis tropicalis is a complex large tubular structure consisting of two halves, separate anteriorly and jointed posteriorly. Each half has two long tubes; inner tube into which the vas deferens opens is termed as vesicular seminalis while the outer tube is termed as accessory tube. The epithelium of both the tubes is glandular in nature. The histology of vesicular apparatus suggests that it performs secretory as well as the storage function.

Multi Stage Seaming Process for Large Tubular Mechanical Bonded Structure

This study is dedicated to three-dimensional finite element analysis of seaming process, which consists of bending, curling and caulking process, of a large tubular mechanical bonded structure. The seaming process is often used to improve a high bonding strength as avoiding any kind of defect. Finite element simulations of the seaming process were preformed for two different initial conditions with pre-analyzed results and without those from bending process. The mechanical bonding strength of the seamed area in the large tubular structure was estimated and compared through finite element analysis among several different analysis conditions of the bending and the caulking. Tensile test for the specimen extracted from the large tubular mechanical bonded structure was also executed and compared with the results of finite element analysis, in order to verify which initial condition in finite element analysis was suitable for this kind of multi stage seaming process. As a result, the effect on an accuracy of finite element analysis for the multi stage seaming process was evaluated in this study. Finally, it is noted that the pre-analyzed results from bending process should be considered in order to obtain the accurate results from finite element analysis.

Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography

Barth syndrome (BTHS) is a mitochondrial disorder that is caused by mutations in the tafazzin gene, which affects phospholipid composition. To determine whether this defect leads to alterations in the internal three-dimensional organization of mitochondrial membranes, we applied electron microscopic tomography to lymphoblast mitochondria from BTHS patients and controls. Tomograms were formed from 50 and 150 nm sections of chemically fixed lymphoblasts and the data were used to manually segment volumes of relevant structural details. Normal lymphoblast mitochondria contained well-aligned, lamellar cristae with slot-like junctions to the inner boundary membrane. In BTHS, mitochondrial size was more variable and the total mitochondrial volume per cell increased mainly due to clusters of fragmented mitochondria inside nuclear invaginations. However, mitochondria showed reduced cristae density, less cristae alignment, and inhomogeneous cristae distribution. Three-dimensional reconstruction of BTHS mitochondria revealed zones of adhesion of the opposing inner membranes, causing obliteration of the intracrista space. We found small isolated patches of adhesion as well as extended adhesion zones, resulting in sheets of collapsed cristae packaged in multiple concentric layers. We also found large tubular structures (diameter 30–150 nm) that appeared to be derivatives of the adhesion zones. The data suggest that mitochondrial abnormalities of BTHS involve adhesions of inner mitochondrial membranes with subsequent collapse of the intracristae space.

Misexpression of acetylcholinesterases in the C. elegans pha-2 mutant accompanies ultrastructural defects in pharyngeal muscle cells

pha-2 is the Caenorhabditis elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here, we performed a detailed characterization of the pha-2 phenotype using cell-type-specific reporters, physical manipulation of the nuclei in pharyngeal muscle cells using “optical tweezers”, electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are (i) the pha-2 ( ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) in the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker than in normal cells and is severely disrupted by large tubular structures and organelles; (iii) the pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) the anc-1 mutant with mislocalized pm5 cell nuclei occasionally shows an isthmus phenotype similar to that of pha-2 worms.

URETERAL DUPLICATION IN A DOG

A male neutered dog of unknown age had recurrent urinary tract infection and caudal abdominal pain. Using sonography, large dilated tubular structure filled with echogenic fluid was seen extending from the left kidney to the level of the bladder neck. In an excretory urogram there was left hydronephrosis with a normal ureter. Computed tomographic evaluation of the abdomen confirmed a large tubular structure extending from the kidney with a blind ending caudally. The left kidney, ureter, and associated tubular structure were surgically removed. No connection was found between the tubular structure and the ureter. Gross evaluation and histopathologic evaluation confirmed the tubular structure to be a ureter, consistent with a diagnosis of ureteral duplication.

Smart Hydrogels for in Situ Generated Implants

The objective of this study was to explore the use of reverse thermo-responsive (RTG) polymers for generating implants at their site of performance, following minimally invasive surgical procedures. Aiming at combining syringability and enhanced mechanical properties, a new family of injectable RTG-displaying polymers that exhibit improved mechanical properties was created, following two different strategies: (1) to synthesize high-molecular-weight polymers by covalenty joining poly(ethylene glycol) and poly(propylene glycol) chains using phosgene as the coupling molecule and (2) to cross-link poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblocks after end-capping them with triethoxysilane or methacrylate reactive groups. While the methacrylates cross-linked rapidly, the triethoxysilane groups enabled the system to cross-link gradually over time. The chain-extended PEO/PPO copolymers had molecular weights in the 39 000-54 000 interval and exhibited improved mechanical properties. Reverse thermo-responsive systems displaying gradually increasing mechanical properties were generated by cross-linking triethoxysilane-capped (EO)(99)-(PO)(67)-(EO)(99) (F127) triblocks. Over time, the ethoxysilane groups hydrolyzed and created silanol moieties that subsequently condensated. With the aim of further improving their mechanical behavior, F127 triblocks were reacted with methacryloyl chloride and the resulting dimethacrylate was subsequently cross-linked in an aqueous solution at 37 degrees C. The effect of the concentration of the F127 dimethacrylate on the mechanical properties and the porous structure of the cross-linked matrixes produced was assessed. Rheometric studies revealed that the cross-linked hydrogels attained remarkable mechanical properties and allowed the engineering of robust macroscopic constructs, such as large tubular structures. The microporosity of the matrixes produced was studied by scanning electron microscopy. Monolayered conduits as well as structures comprising two and three layers were engineered in vitro, and their compliance and burst strength were determined.

The cauliflower mosaic virus capsid protein: assembly and nucleic acid binding in vitro.

The capsid protein of the cauliflower mosaic virus (CaMV) was expressed in a bacterial system to study CaMV assembly. Bacterial lysates contained soluble particulate material and insoluble inclusion bodies that were both used for analysis. In vitro renaturation of pIV derivatives lead to the appearance of folded sheets or large tubular structures in electron microscopy. The region between amino acid positions 77 and 332 is sufficient for self-aggregation of pIV in vitro. C-terminal deletion to amino acid position 265 still allowed dimerization but prevented further aggregation. Nucleic acid binding assays of immobilized pIV derivatives demonstrated that a region located upstream of the retroviral "zinc finger-like" motif is involved in unspecific binding dsDNA, ssDNA and RNA.

Brefeldin A enables synthesis of active lipoprotein lipase in cld/cld and castanospermine-treated mouse brown adipocytes via translocation of Golgi components to endoplasmic reticulum.

Brown adipocytes cultured from newborn combined-lipase-deficient (cld/cld) mice and castanospermine (CST)-treated 3T3-L1 adipocytes synthesize lipoprotein lipase (LPL) which is inactive and retained in the endoplasmic reticulum (ER) [Masuno, Blanchette-Mackie, Chernick and Scow (1990) J.Biol. Chem. 265, 1628-1638; Masuno, Blanchette-Mackie, Schultz, Spaeth, Scow and Okuda (1992) J. Lipid Res.33, 1343-1349]. Brefeldin A (BFA), which is known to block protein transport from ER and translocate Golgi components to ER, was used here to study the effect of translocated Golgi enzymes on LPL retained in ER of cld/cld and CST-treated mouse brown adipocytes. Brown adipocytes cultured from newborn normal mice contained 3000-5000 m-units of LPL activity/mg of DNA and secreted 35 m-units of LPL activity/mg of DNA per h. BFA at 10 micrograms/ml doubled LPL activity in normal cells within 2 h as it stopped completely secretion of active LPL. LPL in mouse cells has two N-oligosaccharide chains per subunit. Analyses with SDS/PAGE and immunoblotting showed that about one-third of LPL subunits in untreated normal cells were totally endo-beta-N-acetylglucosaminidase (endo H)-resistant, one-third were partially endo H-resistant, and one-third were totally endo H-sensitive. BFA decreased to zero the proportion of subunits which were totally endo H-resistant, while it increased the proportion which were partially endo H-resistant. Thus, BFA blocked processing of one oligosaccharide chain per subunit to endo H-resistance. Sucrose-gradient centrifugation studies showed that BFA increased the proportion of LPL subunits in normal cells which were present as active dimers. LPL activity in cld/cld adipocytes was 120 m-units/mg of DNA and that in normal adipocytes treated with CST was 430 m-units/mg of DNA. Most LPL subunits in such cells were totally endo H-sensitive and some were partially endo H-resistant, but none were totally endo H-resistant. Some of the subunits, in both cld/cld and CST-treated cells, were present as inactive LPL dimers. BFA increased LPL activity in cld/cld cells to 2100 m-units/mg of DNA and that in CST-treated cells to 2600 m-units/mg of DNA within 2 h. BFA increased in both groups the proportion of LPL subunits which were partially endo H-resistant. BFA also increased the proportion which were present as active dimers. Immunofluorescence studies in normal and cld/cld adipocytes showed that BFA caused retention of LPL in large tubular and spherical structures and in ER, but not in Golgi. When BFA was withdrawn and protein synthesis was blocked with cycloheximide, LPL in normal cells was transferred to Golgi within 30 min and disappeared within 60 min, whereas LPL in cld/cld cells was retained in large vesicles and ER. The findings indicate that BFA enabled synthesis of active LPL in cld/cld and CST-treated cells via translocation of Golgi components to ER. Also, cld/cld cells synthesized LPL which could be processed to active lipase and the enzymes needed for activation of the lipase were present in Golgi of such cells. Production of inactive LPL in cld/cld adipocytes probably results from their inability to transport LPL from ER to Golgi.

Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord—IV. Effects of peripheral axotomy with special reference to neuropeptide Y and vasoactive intestinal polypeptide/peptide histidine isoleucine

Using immunofluorescence histochemistry and pre- and post-embedding immunoelectron microscopy the rat lumbar dorsal horn was analysed in normal rats and 14 days after unilateral transection of the sciatic nerve. A marked increase in neuropeptide Y-like immunoreactivity was observed in the ipsilateral, superficial dorsal horn, especially in laminae III and IV, of the lumbar 4-5 spinal cord segments after peripheral axotomy. In the ipsilateral lamina II two types of neuropeptide Y-immunoreactive, presumably primary afferent terminals could be identified at the ultrastructural level. The first type contained many large dense-core vesicles (100-155 nm in diameter), whereas a second, more common type had only a few and smaller large dense-core vesicles (80-100 nm in diameter), plus synaptic vesicles of varying diameter (50-85 nm), large empty vesicles and tubular structures. Only occasionally were neuropeptide Y-positive terminals in lamina II involved in the formation of axonal labyrinths. In the ipsilateral lamina III, the number of neuropeptide Y-positive nerve terminals markedly increased after axotomy, with a moderate increase in lamina IV. These neuropeptide Y-positive terminals were morphologically similar to the second type of neuropeptide Y-positive terminal in lamina II, i.e. contained many synaptic vesicles (45-50 nm in diameter), a few small large dense-core vesicles (80-100 nm in diameter), electron-dense granular matrix and a few tubular structures. Fusion of synaptic vesicles with the plasma membrane was often observed at these synapses. These terminals frequently formed glomeruli but were not involved in axonal labyrinths. With regard to local neurons, neuropeptide Y-like immunoreactivity was observed in many dendrite-like profiles mostly making synaptic contacts with neuropeptide Y-negative dendrites and only rarely contacting the central terminal of the glomeruli. Neuropeptide Y-positive nerve endings were mainly seen in lamina I and the outer third of lamina II. After peripheral axotomy the number of vasoactive intestinal polypeptide/peptide histidine isoleucine immunoreactive terminals was increased in laminae I and II. They contained many large dense-core vesicles (100-120 nm in diameter), and some of them were positive for vasoactive intestinal polypeptide/peptide histidine isoleucine. Morphologically, the terminals were characterized by a granular matrix, tubular structures, empty vesicles, reduction in synaptic vesicles and absence of postsynaptic densities. Vasoactive intestinal polypeptide/peptide histidine isoleucine-like immunoreactivities were often found in association with labyrinth formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell tropism and expression of mouse hepatitis viruses (MHV) in mouse spinal cord cultures

Mouse hepatitis viruses (MHV) are coronaviruses which cause various infections in mice affecting lung, intestine, liver, and other organs as well as the central nervous system. The replication of three different MHV strains was studied in mouse dissociated spinal cord cultures containing differentiated neurons and nonneuronal cells (NN) (including astrocytes). Cell tropism and maturation of each virus strain was analyzed by immunolabeling methods using antisera to the virion or to purified membrane glycoproteins (E 1 and E 2) and by electron microscopy (EM). Wt-JHM, which causes acute encephalitis in mice, produces acute cytopathic changes in both neurons and NN cells. In neurons, virions mature in smooth ER cisternae closely associated to the Golgi apparatus. As judged by EM, fewer virions are produced by neurons than NN cells and neurons do not fuse or stain for E 2 as do NN cells. NN cells contain large inclusions made of nucleocapsid strands. A temperature-sensitive mutant of JHM, Ts8-JHM, which causes demyelination in mice, infects NN cells but not neurons. Infected NN cells synthesize E 1 and E 2, and contain large inclusions but few mature virions, even at permissive temperatures. These inclusions appear granular and rarely contain nucleocapsid strands in contrast to wt-JHM infection. NN cells infected with this mutant also display numerous membrane whorls. The hepatotropic strain A59 lacks tropism for neurons and primarily infects NN cells, thus resembling ts8-JHM. Infected NN cells become loaded with intracytoplasmic virions which are secreted from the cells. E 1 can only be detected in the perinuclear area of these cells while E 2 rapidly spreads throughout the cytoplasm. The cytoplasm of A59 infected NN cells frequently contains large tubular structures often in the lumen of the RER. In conclusion, in primary CNS cultures consisting of neurons and NN cells: (1) wt-JHM replicates in both neurons and NN cells but has different effects on these cells; (2) Ts8-JHM exhibits no productive infection of neurons, and in NN cells appears to be defective in assembly and to stimulate membrane synthesis; (3) A59 also shows tropism restricted to NN cells which produce many viruses and display differential distribution of the two virion glycoproteins. Thus, in the absence of the immune system, the MHV strains assayed exhibit differences in viral tropism, cytopathic changes, and viral assembly in CNS cells, and these differences may account for the different disease patterns.

Assembly of bacteriophage T4 head-related structures: IV. Isolation and association properties of T4 prehead proteins

The following bacteriophage T4 prehead proteins have been purified by preparative isoelectrofocusing in a layer of Sephadex: (1) P22, the major protein of the prehead core; (2) P23, the major protein of the prehead shell; (3) P24, a minor shell protein; (4) a mixture of IPIII and IPII, two alkaline core proteins; and (5) a protein complex containing several core proteins. We have identified distinct association states in which these prehead proteins occur. Analytical ultracentrifugation, electrophoresis of crosslinked oligomeric protein complexes and electron microscopy have been used. Under mild prehead-dissociating conditions the proteins are predominantly monomeric. Under conditions that promote prehead assembly in vitro prehead proteins occur in the following association states: (1) P22 forms a small oligomer and long, very flexible filamentous structures of about 9 nm wide; (2) P23 associates into a hexamer and into large tubular structures which have a hexagonal surface lattice (polyheads); (3) P24 forms pentamers, no large structures have been found; (4) IPIII and IPII remain monomeric; and (5) several core proteins together form a complex with a molecular weight of the order of 200,000. These results allow conclusions about the building principle and assembly mechanism of the T4 prehead. The prehead seems to consist of two different polymeric structures. One is the P22 filament, which probably forms the backbone of the prehead core; the other is the hexagonal P23 lattice of the prehead shell. These structures constitute a framework to which other, accessory proteins, like IPIII in the core and P24 in the shell, can attach.