Base Composition and Hybridization Studies of the Three Double-Stranded RNA Segments of Bacteriophage φ6

The three double-stranded ribonucleic acid (dsRNA) segments of the bacteriophage phi6 were isolated and shown to have similar melting temperatures and base compositions. RNA:RNA hybridization experiments with the isolated segments eliminate the possibility that the two smaller dsRNA segments arise from a cleavage of the large dsRNA segment. The two smaller RNA segments reanneal rapidly even at low temperatures; in contrast, the large dsRNA reannealed only at higher temperatures. Evidence is also presented which suggests that the dsRNAs may contain a short single-stranded RNA tail.     

Mechanism of Adsorption and Eclipse of Bacteriophage φX174 III. Comparison of the Activation Parameters for the In Vitro and In Vivo Eclipse Reactions With Mutant and Wild-Type Virus

In a starvation buffer containing 10(-3) M divalent cations, phiX174 undergoes viral eclipse above 20 C when attached to intact host cells. An in vitro structural transition that is similar to that observed in this in vivo eclipse reaction occurs over the same temperature range in 0.1 M CaCl(2) (pH 7.2). Since both reactions result in a loss of infectivity, their kinetics have been compared in this report. Both exhibit a biphasic first-order loss in PFU that is a result of two competing first-order processes. However, a single type of heterogeneity in the population of virions is not the basis for both competing slower reactions. The Arrhenius plots of the faster components show that the in vitro eclipse reaction has the same activation energy of 35 kcal/mol (ca. 1.47 x 10(5) J/mol) as the in vivo reaction but a 10-fold lower Arrhenius preexponential factor. This is further evidence that certain features of the in vivo mechanism are retained in the in vitro reaction. In the case of the slower components, the in vitro reaction has an activation energy of 37 kcal/mol (1.55 x 10(5) J/mol), whereas that of the in vivo reaction is only 5 kcal/mol (2.1 x 10(4) J/mol). A similar analysis has been performed on a cold-sensitive eclipse mutant of phiX174. In vivo, the mutation is expressed by a two- to three-fold lower Arrhenius preexponential factor for both components of the eclipse reaction when compared to wt virus. The activation energies for both components are the same as wt virus. These results suggest that the mechanism of the eclipse reaction can be operationally divided into two aspects, each subject to mutational alteration.     

Studies on the Biosynthesis of α-Putrescinylthymine in Bacteriophage φW-14-Infected Pseudomonas acidovorans

The alpha-putrescinylthymine (putThy) in bacteriophage phiW-14 DNA is synthesized at the mononucleotide level: it is labeled by uracil or deoxyuridine but not by thymidine, and it appears in the acid-soluble pool of infected cells before the onset of phage DNA synthesis. The methylene group at the C-5 position of the pyrimidine moiety of putThy is derived in vivo from a C(1) unit. Extracts of a phage infected thymidine auxotroph of the host, Pseudomonas acidovorans, apparently contain a phage-specific thymidylate synthetase and a phage-specific activity which forms 5-hydroxymethyl dUMP from N(5), N(10)-methylene-tetrahydrofolate and dUMP.     

Involvement of a Tryptophan Residue in the Assembly of Bacteriophages φ80 and Lambda

The assembly of infective particles of bacteriophages lambda and phi80 from heads and tails was found to be inhibited by l-tryptophan and some of its analogues, most notably tryptamine. Both the rate of assembly and the final yield of phage were inhibited. The amino acid l-phenylalanine had a slight inhibitory effect, whereas all other amino acids found in proteins were ineffective. Evidence was presented to show that the binding of heads to tails was the affected process in the assay for assembly of infective units. The plaque-forming ability of preassembled phage was not affected by these inhibitors. Results of three different types of experiments suggest that inhibition is due to interaction of inhibitors with the head substructure. The assembly reaction is highly dependent on pH, ionic strength, and the presence of detergents.     

Mechanism of Adsorption and Eclipse of Bacteriophage φX174 I. In Vitro Conformational Change Under Conditions of Eclipse

Bacteriophage phiX174 undergoes a conformational change during viral eclipse when virus-host cell complexes are incubated briefly at 37 C in a complex starvation buffer at pH 8. In this report, basically the same transition is demonstrated in vitro. Incubation of phiX alone for 2 to 3 hr at 35 C in 0.1 m CaCl(2) (pH 7.2) results in an irreversible decrease in S(20,w) because of an increase in the frictional coefficient that occurs during the change in conformation. The slower sedimenting conformation is noninfectious. These properties are remarkably similar to those of the eclipsed particles characterized by Newbold and Sinsheimer. Therefore, the key structural requirements for the molecular mechanism must reside within the architecture of the virus itself. This extremely simplified system uncovered the calcium ion requirement and pronounced dependence on pH between 6 and 7, both inherent properties of adsorption. This and the more than 10-fold greater rate of the in vivo conformational transition allude to the cooperative nature of attachment and eclipse for phiX.     

Morphogenesis of Bacteriophage φ80: Identification of the Cistron 13 Product

An in vitro complementation reaction leading to the assembly of bacteriophage phi80 tails from component proteins is described. Tail assembly occurs when a lysate of any mutant in cistron 13 is mixed with a second lysate of a mutant in any of the other cistrons involved in tail formation. Lysates of mutants that are blocked in tail formation contain phage heads that can unite with free tails to form infective particles. The rate of the complementation reaction shows little dependence upon temperature, suggesting that the assembly depends largely upon the kinetic encounter of the interacting components. The tail component missing in cistron 13 mutant lysates was purified approximately 55-fold and shown to be, at least in part, a protein having a molecular weight of approximately 22,000. This protein was also released from highly purified infective phi80 particles after osmotic shock followed by heattreatment, suggesting that it most probably is an integral structural protein of the phage tail. Lysates of mutants of bacteriophage lambda that are defective in tail formation were shown to contain a tail component identical with or similar to the phi80 cistron 13 product.     

Effects of Furazolidone on the Infection of Vibrio cholerae by Bacteriophage φ149

Furazolidone in concentrations which had little effect on the growth of host organisms greatly reduced the yield of phage 149 from the host Vibrio cholerae OGAWA 154. This phage was resistant to the in vitro action of the drug. The phage yield of infected bacteria depended significantly on the time of addition or withdrawal of the drug. The average burst size of the drug-treated and infected bacteria decreased exponentially with increase in drug concentration. The latent period of phage multiplication and also the eclipse period did not change significantly from the control values. A concentration of 0.05 μg of furazolidone per ml inhibited DNA synthesis by about 50% in phage-infected cells and only by about 18% in noninfected ones, relative to the respective controls. RNA and protein synthesis were affected by a much smaller degree both in infected and noninfected cells. Quantitative deduction of the length of furazolidone-treated cells from their phage adsorption characteristics and its agreement with previous electron microscopy data indicated that furazolidone did not affect the phage receptors.     

Location of the 5-Methylcytosine Group on the Bacteriophage φX174 Genome

Bacteriophage phiX174 DNA was labeled in vivo with [methyl-(3)H]methionine. The methyl-labeled progeny DNA was extracted from purified bacteriophage phiX174 particles and was used as template for in vitro synthesis of the complementary strand in the presence of the nucleoside triphosphates and Escherichia coli polymerase I. The resultant replicative form DNA was then cleaved, in separate experiments, with restriction endonucleases from Haemophilus influenzae and H. aegyptius. The DNA fragments were analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methylcytosine in the viral DNA is located in a specific region of the phiX174 genome, very likely in gene H.     

The Structure of Bacteriophage φCb5 Reveals a Role of the RNA Genome and Metal Ions in Particle Stability and Assembly

The structure of the Leviviridae bacteriophage φCb5 virus-like particle has been determined at 2.9 Å resolution and the structure of the native bacteriophage φCb5 at 3.6 Å. The structures of the coat protein shell appear to be identical, while differences are found in the organization of the density corresponding to the RNA. The capsid is built of coat protein dimers and in shape corresponds to a truncated icosahedron with T = 3 quasi-symmetry. The capsid is stabilized by four calcium ions per icosahedral asymmetric unit. One is located at the symmetry axis relating the quasi-3-fold related subunits and is part of an elaborate network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilize the contacts within the coat protein dimer. The stability of the φCb5 particles decreases when calcium ions are chelated with EDTA. In contrast to other leviviruses, φCb5 particles are destabilized in solution with elevated salt concentration. The model of the φCb5 capsid provides an explanation of the salt-induced destabilization of φCb5, since hydrogen bonds, salt bridges and calcium ions have important roles in the intersubunit interactions.Electron density of three putative RNA nucleotides per icosahedral asymmetric unit has been observed in the φCb5 structure. The nucleotides mediate contacts between the two subunits forming a dimer and a third subunit in another dimer. We suggest a model for φCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The φCb5 structure is the first example in the levivirus family that provides insight into the mechanism by which the genome-coat protein interaction may accelerate the capsid assembly and increase capsid stability.     

Morphology and Physiology of the Intracellular Development of Bacillus subtilis Bacteriophage φ25

The morphology of the intracellular development of bacteriophage phi25 in Bacillus subtilis 168M has been correlated with nucleic acid synthesis in infected cells. Host deoxyribonucleic acid (DNA) synthesis was shut off by a phage-induced enzyme within 5 min after infection, and another phage-mediated function extensively degraded host DNA at the time of cell lysis. Synthesis of phage DNA in infected cells began within 5 min and continued until late in the rise period. After phage DNA synthesis and coinciding with lysis, much of the unpackaged, newly synthesized phage DNA was degraded. Studies of thin sections of phi25 infected cells suggested that unfilled capsids may be precursors to filled capsids in the packaging process. To assess dependence of capsid formation on phage DNA replication, cells were either treated with mitomycin C and infected with normal phage or infected with ultraviolet-irradiated (99% killed) phi25. Only empty capsids were found in these cells, indicating that capsid production may be independent of the presence of newly synthesized viral DNA.     

Structure of Bacillus subtilis Bacteriophage φ25 and φ25 Deoxyribonucleic Acid

The structure of Bacillus subtilis bacteriophage phi25 and phi25 deoxyribonucleic acid (DNA) were studied by electron microscopy. The head of phi25 is a regular polyhedron measuring 75 nm in diameter. The uncontracted tail of phi25 is 130 nm in length and includes a large, complex tail plate. Phage phi25 DNA is double-stranded and has a molecular weight of approximately 100 million as determined by electron microscopic length measurements and analytical band sedimentation in CsCl. The complementary strands of phi25 DNA contain numerous random interruptions. Chemical analysis of phi25 DNA demonstrated that 5-hydroxymethyluracil replaces thymine and that the DNA has a mole per cent (guanine plus cytosine) of 42.     

Genetic Study of Suppressor-Sensitive Mutants of the Bacillus subtilis Bacteriophage φ29

With bacteriophage phi29 of Bacillus subtilis 133, suppressor-sensitive (sus) hydroxylamine mutants have been isolated. Intracistronic and intercistronic quantitative complementation placed the mutants in 13 cistrons, and three-factor crosses have been used to assign an unambiguous order for 10 cistrons. Recombination frequencies have been presented for several regions of the genome to facilitate comparison of the sus system with the previously published temperature-sensitive mapping systems.     

Synthesis of Novel Quinoline–Benzoxazolinone Ester Hybrids: In Vitro Anti-Inflammatory Activity and Antibacterial Activity

Russian Journal of Bioorganic Chemistry - A series of novel quinoline-benzoxazolinone ester hybrids were synthesized characterized and assessed for their in vitro anti-inflammatory and...     

Influence of Guar Gum on the In Vitro Starch Digestibility—Rheological and Microstructural Characteristics

The present study investigates the effect of guar gum on the digestibility of a waxy maize starch in vitro under simulated gastric and intestinal conditions. A detailed rheology and confocal scanning laser microscopy of the digesta were performed in order to study the possible mechanisms of interactions involved during in vitro hydrolysis of starch. No starch hydrolysis was observed under simulated gastric conditions, whereas more than 90% hydrolysis was observed at the end of digestion under simulated intestinal conditions. In the presence of guar gum, the starch hydrolysis was reduced by nearly 25% during the first 10 min and by 15% at the end of in vitro intestinal digestion. The rheological behavior of the digesta was significantly affected in the presence of the gum. The viscosity of digesta decreased during intestinal digestion; however, the extent of decrease was quite low in the presence of guar gum. The consistency index increased and flow behavior index of digesta decreased in the presence of gum after 1 min of intestinal digestion. All the samples (digested or undigested) displayed a pseudoplastic behavior as their apparent viscosity (η _a) decreased with an increase in shear rate. A negative correlation between the starch hydrolysis (%) and storage modulus for the starch sample without gum and a positive correlation for the starch sample with gum were found. Large granule remnants observed through confocal micrographs have shown that the solubilization of starch granule remnants during in vitro digestion is significantly reduced in the presence of gum.     

Studies with Bacteriophage φII. Events Following Infection of Male and Female Derivatives of Escherichia coli K-12

We studied the course of infection of the female-specific bacteriophage phiII in male and female cells isogenic except for the presence of the substituted sex factor, F'lac. Both male and female cells are killed by phiII; however, only limited phage replication occurs in male cells. Host macromolecular synthesis stops abruptly at 4 to 6 min after infection of male cells, and synthesis of phage components cannot be detected. Experiments with chloramphenicol indicate that phage deoxyribonucleic acid (DNA) penetrates into male cells, since protein synthesis after infection is required to stop synthesis of DNA in males. Phage DNA becomes membrane-associated in both female and male cells. In male cells, parental phage DNA does not dissociate from the membrane during the latent period as is the case with females, indicating a block in phage DNA replication. Isolation of nonrestricting F'lac mutations indicates involvement of a specific episome product in phiII restriction.     

Effects of Oxygen Concentration on the Proliferation and Differentiation of Mouse Neural Stem Cells In Vitro

Background and purpose Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro. Methods NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively. Results The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions. Conclusions These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.     

Effects of Mutations and Post-Translational Modifications on α-Synuclein In Vitro Aggregation

Fibrillar aggregates of the α-synuclein (αS) protein are the hallmark of Parkinson’s Disease and related neurodegenerative disorders. Characterization of the effects of mutations and post-translational modifications (PTMs) on the αS aggregation rate can provide insight into the mechanism of fibril formation, which remains elusive in spite of intense study. A comprehensive collection (375 examples) of mutant and PTM aggregation rate data measured using the fluorescent probe thioflavin T is presented, as well as a summary of the effects of fluorescent labeling on αS aggregation (20 examples). A curated set of 131 single mutant de novo aggregation experiments are normalized to wild type controls and analyzed in terms of structural data for the monomer and fibrillar forms of αS. These tabulated data serve as a resource to the community to help in interpretation of aggregation experiments and to potentially be used as inputs for computational models of aggregation.     

Is the In?Vitro Ejection of Bacteriophage DNA Quasistatic? A Bulk to Single Virus Study

Bacteriophage T5 DNA ejection is a complex process that occurs on several timescales in vitro. By using a combination of bulk and single phage measurements, we quantitatively study the three steps of the ejection—binding to the host receptor, channel-opening, and DNA release. Each step is separately addressed and its kinetics parameters evaluated. We reconstruct the bulk kinetics from the distribution of single phage events by following individual DNA molecules with unprecedented time resolution. We show that, at the single phage level, the ejection kinetics of the DNA happens by rapid transient bursts that are not correlated to any genome sequence defects. We speculate that these transient pauses are due to local phase transitions of the DNA inside the capsid. We predict that such pauses should be seen for other phages with similar DNA packing ratios.