Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA 2.

The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.     

Characterization of IS 1110, a highly mobiie genetic element from Mycobacterium avium

A highly mobile Insertion sequence designated IS 1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20′) were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS 1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS 1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited In M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.     

City refuse compost as a phosphorus source to overcome the P-fixation capacity of sesquioxide-rich soils

In sesquioxide-rich soils of tropical and subtropical areas and volcanic-ash soils with high levels of active Al(Fe), large amounts of phosphate fertilizers are needed to overcome their high P-fixation capacity (quenching strategy). A greenhouse pot experiment has been used to evaluate the effectiveness of city refuse compost (CRC) as a P-source for these variable-charge soils, compared to inorganic P. Mature CRC and K₂HPO₄ were applied at rates equivalent to 125, 250, 375, 500 and 625 kg P ha^–1 to a ferrallitic soils from Tenerife Island (Andeptic Paleudult) with a high content in active Al+Fe (4.82%) and a high P-fixation capacity (87%). Perennial ryegrass (Lolium perenne L.) was grown in pots and plants were harvested at regular intervals after seedling emergence. CRC increases plant P concentration and soil labile-P proportional to the applied rate. The best results were obtained from a compost application of 30 t ha^–1 equivalent-rate, after a residence time of at least three months. An important residual effect in the supply capacity of P in relation to the phosphate fertilizer was also observed. The relative agronomic effectiveness (RAE) in comparison to K₂HPO₄ was 66% after 6 months, considering P uptake + soil labile-P. The soil P-fixation capacity was significantly reduced from a compost application of 40 t ha^–1 equivalent-rate. Competition in adsorption between organic ligands and phosphate, in combination with net mineralization of organic P in compost, might account for the high RAE value obtained. The main conclusion is that the city refuse compost could be a suitable P-amendment for resquioxic soils due to its high RAE, and the residual effect on P-supply. ei]H. Lambers     

Mycoplasma pneumoniae infection and arthritis in man.

Seven patients developed arthritis after Mycoplasma pneumoniae infection. Their joint symptoms persisted for up to one year. One patient developed an articular erosion and in another rheumatoid factor was present in serum transiently. Mycoplasma infection often causes ill-defined arthralgias and myalgias, but the migratory polyarthropathy of middle-sized joints that occurred in these patients is much less common. The prognosis seems to be good.     

Escherichia coli ppGpp synthetase II activity requires spoT

Escherichia coli has two enzymes catalyzing the synthesis of guanosine tetraphosphate (ppGpp), designated ppGpp synthetase I (PSI = RelA) and II (PSII), whose activities are regulated differently. Until now, the gene for PSII had not been identified. Here, an E. coli relA1 strain that expresses lacZ from an rrnB P1 promoter was used to screen mutants with increased beta-galactosidase activity on 5-bromo-4-chloro-3-indoyl beta-D-galactoside indicator plates at 30 degrees C. About 15% of the mutants obtained in this manner had reduced levels of ppGpp at 30 degrees C and no detectable ppGpp at 43 degrees C. These mutants did not form colonies at 42 degrees C on minimal medium plates and had elevated ribosome concentrations and higher growth rates at 30 degrees C. Genetic mapping by phage P1 transduction and complementation analyses showed that the mutations were located in spoT and that they were recessive. Specific inhibition of SpoT-dependent ppGpp degradation activity with picolinic acid showed that two of the mutants tested were deficient in ppGpp synthesis activity. These results indicate that spoT is required for PSII activity, suggesting that spoT encodes both ppGpp degradation and synthesis activities and that these two functions can be affected independently by mutation.     

Grayanotoxin-I-modified eel electroplax sodium channels. Correlation with batrachotoxin and veratridine modifications.

To probe the structure-function relationships of voltage-dependent sodium channels, we have been examining the mechanisms of channel modification by batrachotoxin (BTX), veratridine (VTD), and grayanotoxin-I (GTX), investigating the unifying mechanisms that underlie the diverse modifications of this class of neurotoxins. In this paper, highly purified sodium channel polypeptides from the electric organ of the electric eel were incorporated into planar lipid bilayers in the presence of GTX for comparison with our previous studies of BTX (Recio-Pinto, E., D. S. Duch, S. R. Levinson, and B. W. Urban. 1987. J. Gen. Physiol. 90:375-395) and VTD (Duch, D. S., E. Recio-Pinto, C. Frenkel, S. R. Levinson, and B. W. Urban. 1989. J. Gen. Physiol. 94:813-831) modifications. GTX-modified channels had a single channel conductance of 16 pS. An additional large GTX-modified open state (40-55 pS) was found which occurred in bursts correlated with channel openings and closings. Two voltage-dependent processes controlling the open time of these modified channels were characterized: (a) a concentration-dependent removal of inactivation analogous to VTD-modified channels, and (b) activation gating similar to BTX-modified channels, but occurring at more hyperpolarized potentials. The voltage dependence of removal of inactivation correlated with parallel voltage-dependent changes in the estimated K1/2 of VTD and GTX modifications. Ranking either the single channel conductances or the depolarization required for 50% activation, the same sequence is obtained: unmodified > BTX > GTX > VTD. The efficacy of the toxins as activators follows the same ranking (Catterall, W. A. 1977. J. Biol. Chem. 252:8669-8676).     

Interaction of neurotensin with prostaglandin E2 and prostaglandin synthesis inhibitors: effects on colonic temperature in mice

Neurotensin (NT) administered intracisternally (i.c.) to adult mice produced a marked hypothermia while prostaglandin E₂, administered by the same route, produced hyperthermia. When administered concurrently the effects of the two substances were neutralized. The prostaglandin synthesis inhibitors, indomethacin and acetylsalicylic acid, were injected subcutaneously 30 min prior to i.c. administered NT and/or thyrotropin-releasing hormone (TRH). Both inhibitors failed to potentiate the hypothermia induced by NT or alter its antagonism by TRH in mice kept at 26°C. When mice were kept at 6°C, pretreatment with indomethacin, but not acetylsalicylic acid, potentiated NT-induced hypothermia and prevented its antagonism by TRH. Because indomethacin inhibits synthesis of prostaglandins within the central nervous system (CNS) as well as in peripheral organs while acetylsalicylic acid acts only in the periphery, it appears that NT-induced hypothermia in a cold environment is enhanced by a reduction of prostaglandins in the CNS.     

Synthesis and relative stereochemistry of the four mercapturic acids derived from styrene oxide and N-acetylcysteine

The chemical reaction between (±)-styrene oxide and N-acetylcysteine produces both positional isomers ($\text{1}$ and $\text{2}$) as a mixture of diastereoisomers with a preference for the benzylic thioether isomer $\text{1}$ (2 : 1). Synthesis of the mercapturic acid conjugates from either (+)- or (?)-styrene oxide produces only two of the four possible stereoisomers. The single diastereoisomers of $\text{1}$ and $\text{2}$ were separated by high pressure liquid chromatography (HPLC) and identified by ¹H- and ¹³C-nuclear magnetic resonance (NMR). The relative stereochemistry at the benzylic carbon center of the mercapturic acid conjugates was assigned on the basis of the established chemical correlation between optically pure styrene oxide and its precursor mandelic acid, and considerations on the mechanism of ring opening of epoxides by sulfur nucleophiles. The stereochemical definition of the isomers $\text{3}$–$\text{6}$ should prove useful in investigations of the biotransformation of the glutathione (GSH) conjugates of styrene oxide.