Cytoplasmic helical structure associated with Acholeplasma laidlawii.

A distinct spiral protein structure was found in three species of Acholeplasma, but was not found in the Mycoplasma species studied. The spirals, which are 14 nm in width and of variable length from 50 to 300 nm, are formed by a helical arrangement of 7-nm subunits. A rosette-like structure 45 nm in diameter also composed of 7-nm subunits was found in close association with the spirals and may be a taut in vivo form of the spiral. The electrophoretic profile in sodium dodecyl sulfate-polyacrylamide gels indicated that the spirals are composed of a predominant polypeptide with an apparent molecular weight of 100,000. No evidence can be found for inferring actin-like properties for this structure.     

Inhibition of HIV-1 replication by a tricyclic coumarin GUT-70 in acutely and chronically infected cells

The anti-HIV-1 activity of GUT-70, a natural product derived from the stem bark of Chlophyllum brasiliense, was evaluated. GUT-70 inhibited HIV-1 replication in both acutely and chronically infected cells through suppression of NF-κB. Our results strengthen the idea that NF-κB pathway is one of the potential targets to control HIV-1 replication and that GUT-70 could serve as a lead compound to develop novel therapeutic agents against HIV-1 infection.     

Comparative study of rosette and plaque formation in rats infected with Mycoplasma arthritidis and Acholeplasma laidlawii]

M. arthritidis suppresses rosette- and plaque-formation in rats infected with this strain of mycoplasma. On the 15th day the immune response was restored in the control and experimental groups of animals. In later stages a strong stimulation of RFC and PFC was recorded, which levelled on day 150. On the contrary, A. laidlawii stimulated RFC and PFC in all stages of infection. A relationship between the phenomena described and pathogenic properties of the mycoplasma is discussed.     

Complete genome and proteome of Acholeplasma laidlawii

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.     

Alternative to fluorescence assays to monitor fusion between Acholeplasma laidlawii cells and liposomes

AIMS: To develop a new technique as an alternative to the fluorescence assays and electron microscopy for the purpose of monitoring the cell-liposome fusion. METHODS AND RESULTS: Acholeplasma laidlawii whole cells did not oxidize Glucose-6-phosphate (G6P) or Fructose-1,6 diphosphate (F1,6DP) as free (unentrapped) substrates, at concentrations 47 and >270 mM, respectively. Lysed A. laidlawii cells oxidized G6P and F1,6DP at lower concentration of 0.8 and 15 mM, respectively. When these substrates were entrapped inside liposomes, at a final concentration of 1.5 mM, and interacted with A. laidlawii whole cells, in an oxygen electrode chamber, an increase in oxygen uptake was evident. This interaction does not have any effect on cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental system described here is advantageous over classical fluorescence assays in determining the fate of liposome-entrapped material and raises the possibility of studying the kinetics of metabolic substrates, which are normally excluded from the cell by the cell membrane.     

Alteration of colonial morphology of Acholeplasma laidlawii and Acholeplasma modicum by infection with Mycoplasmatales viruses.

Morphologically aberrant colonies resulted from the infection of Acholeplasma laidlawii with two of its three known viruses and from Acholeplasma modicum cells naturally carrying virus. The patterns of colonial alteration differed between cells infected with the two A. laidlawii viruses. Colonies derived from single cells infected with the bullet-shaped virus MV-L1 (Mycoplasmatales virus-laidlawii-1) had a radial sectoring pattern of intracolonial swellings ("blebs"), whereas cells infected with the tailed icosahedral virus MV-L3 contained bubble-like blebs. Colonies from cellsinfected with the enveloped virus MV-L2 appeared identical to those obrained from uninfected cells. Aberrant colonies contained 10(6) colony-forming units of organisms and 10(6) plaque-forming units of virus serologically identical to the infecting type, indicating that both the virus and host organism were capable of simultaneous replication. Enumeration of virus by means of counting aberrant colonies was 30-fold more sensitive than infectious center assay for MV-L1 and 1.2- to 2-fold higher for MV-L3. Furthermore, blebbed colonies plaquing with a new virus specific to A. modicum. Thus, blebbing in colonies provides a valuable marker for detection of the Mycoplasmatales viruses.     

Proteins of bacterial membranes. H+-adenosinetriphosphatase from Acholeplasma laidlawii cells

The cytoplasmic membrane of micoplasmic cells, in particular of A. laidlawii cells, contains a proton-carrier Mg2+ -activated ATPase. A whole H+ -ATPase complex (F0-F1) was isolated from these cells and characterized. The isolation procedure included solubilization of the enzyme with Triton X-100 followed by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The enzyme was inhibited by dicyclohexylcarbodiimide (10(-4) M). The Km value for ATP hydrolysis and Ki for ADP hydrolysis were determined. The order of the constants did not differ from those measured earlier for factor F1 of the complex. The purified enzyme, similar to its hydrophylic moiety is sensitive to the action of bivalent cations. The subunit composition of the whole complex and of its water-soluble part was investigated. The complex was found to contain 11 polypeptides, five of which belong to factor F1. The molecular weights of these polypeptides were determined.     

RNA interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.     

Osmotic behaviour of Acholeplasma laidlawii B cells with membrane lipids in liquid-crystalline and gel state.

The osmotic behaviour of Acholeplasma laidlawii B cells was investigated with combined spectrophotometric and enzymatic measurements. The conclusion could be drawn that this osmotic behaviour depends largely on the physical state of the membrane lipids. When part of the membrane lipids is in the liquid-crystalline phase the cell is able to swell and behaves as a good osmometer. However, when the membrane lipid is in the gel phase, the cell is unable to swell and the change in absorbance of the cell suspension is then completely due to lysis.     

Electroporation-mediated transfection of Acholeplasma laidlawii with mycoplasma virus L1 and L3 DNA.

In contrast to mycoplasma virus L1 and L2 circular DNA, mycoplasma virus L3 linear DNA is not biologically active in polyethylene glycol-mediated transfection. Electroporation of Acholeplasma laidlawii, however, leads to plaque formation after incubation with L3 DNA. The efficiency of electroporation-mediated transfection is 1/10 that of polyethylene glycol-mediated transfection as estimated with L1 DNA. Trypsin treatment of cells before DNA addition increases the efficiency of DNA uptake.     

Membrane composition and virus susceptibility of Acholeplasma laidlawii.

The membrane composition of 11 strains of Acholeplasma laidlawii, including three strains persistently infected with mycoplasmaviruses MVL51, MVL2, and MVL3, was studied and correlated with mycoplasmavirus sensitivity. Membranes of the strains had similiar sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, and all strains were inhibited by an antiserum produced against membranes from one of the strains. The amounts of integral membrane proteins solubilized by the nonionic detergent Tween 20 differed considerably. Therefore, characteristic crossed immunoelectrophoresis patterns were obtained for each strain. Strains persistently infected with MVL2 and MVL3 were notably different from the noninfected host. The ability to propagate any of the viruses was not correlated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis or crossed immunoelectrophoresis patterns. The persistently infected strains had a characteristic lipid composition. MVL51-resistant strains, including a resistant clone selected from a sensitive strain, were characterized by a large monoglucosyldiglyceride/diglucosyldiglyceride ratio and trace amounts of diphosphatidylglyceol (as opposed to the sensitive strains). Differences in lipid composition in A. laidlawii seem to affect the relationship between cells and viruses.     

Purification and Properties of Acetate Kinase from Acholeplasma laidlawii

Acetate kinase (EC 2.7.2.1) was purified from Acholeplasma laidlawii cytoplasm by a combination of ammonium sulfate fractionation, gel filtration, diethylaminoethyl-cellulose chromatography, and affinity chromatography on 8-(6-aminohexylamino)-adenosine 5'-triphosphate conjugated to Sepharose 4B. The enzyme was composed of polypeptide chains of about 50,000 molecular weight as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturating conditions, apparent molecular weights between 64,000 and 130,000 were obtained, depending upon mainly the ionic strength of the test solution. The enzyme had a narrow specificity for phosphate acceptor acids, whereas both purine and pyrimidine nucleoside triphosphates were suitable phosphate donors. Na(+) and K(+) inhibited both acetyl phosphate and adenosine 5'-triphosphate synthesis, and the latter was also inhibited by high concentrations of adenosine 5'-diphosphate and acetyl phosphate. This substrate inhibition was partially abolished by 0.5 M NaCl. The enzyme catalyzed the independent adenosine 5'-diphosphate<-->adenosine 5'-triphosphate and acetate<-->acetyl phosphate exchanges. The rate of the latter was enhanced by the addition of cosubstrate Mg(2+)-adenosine 5'-triphosphate. The high affinity for substrates, except for acetate, indicated that under physiological conditions the direction of the enzymic reaction favors adenosine 5'-triphosphate synthesis. Thus, a mechanism for adenosine 5'-triphosphate generation in mycoplasmas is suggested.     

Release of a Group 1 Mycoplasma Virus from Acholeplasma laidlawii After Treatment with Mitomycin C

A rod-shaped group 1 mycoplasma virus was released from Acholeplasma laidlawii strain JA2 after treatment with 2.5 μg of mitomycin C per ml. Similar treatment of A. laidlawii strain Bju failed to stimulate release of any PFU.