Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies.

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Conjugal transfer of group B streptococcal plasmids and comobilization of Escherichia coli-Streptococcus shuttle plasmids to Lactobacillus plantarum.

The antibiotic resistance group B streptococcal plasmids, pIP501 and pVA797, were conjugally transferred from Streptococcus faecalis to Lactobacillus plantarum. The Escherichia coli-Streptococcus shuttle plasmids, pVA838 and pSA3, were mobilized from S. sanguis to L. plantarum by pVA797 via cointegrate formation. pVA838 readily resolved from pVA797 and was present in L. plantarum as deletion derivatives. The pVA797::pSA3 cointegrate failed to resolve in L. plantarum.     

Kelthane degradation by genetically engineered Pseudomonas aeruginosa BS827 in a soil ecosystem.

The aim of this study was to study the degradation of kelthane by Pseudomonas aeruginosa BS827, which carried the plasmid pBS3. This plasmid encodes naphthalene oxidation. The strain was able to survive in the presence of kelthane and to retain its degradative ability. Kelthane also stabilized the biodegradative plasmid that was preserved by 70 to 100% of the cell population. Cells deficient in Nah or Sal characters were less effective in degrading kelthane, whereas plasmid-free cells lost this ability completely. Evidently, the degradative activity of P. aeruginosa BS827 was conditioned by plasmid determinants coupled with genes of the plasmid pBS3 Nah region.     

Degradation of DNA in cells and extracts of the obligately anaerobic bacterium Roseburia cecicola upon exposure to air.

High-molecular-weight chromosomal DNA from Roseburia cecicola, an oxygen-intolerant anaerobe, could be isolated only when the bacterial cells were kept under anaerobic conditions up to the time of cell lysis. When the cells were exposed to oxygen before lysis, the chromosomal DNA degraded. Likewise, linear but not covalently closed circular DNAs degraded in cell extracts of the organism that were exposed to atmospheres containing O2 but not in extracts that were maintained in a reduced state. Covalently closed circular DNAs were nicked but not degraded in the oxidized extracts.     

Presence of aflatoxin M1 in commercial ultra-high-temperature-treated milk.

Forty-seven samples of commercial ultra-high-temperature-treated milk from a dairy facility in the northwest part of Spain were analyzed for the presence of aflatoxin M1. A total of 14 samples (29.8%) were positive for aflatoxin M1 (4 in May, 3 in November, 3 in December, 1 in January, 1 in April, 1 in July, and 1 in August), 29 (61.7%) were negative, and 4 (8.5%) were doubtful, i.e., they showed trace quantities of aflatoxin M1. The range of aflatoxin M1 content was 0.02 to 0.1 ng/ml.     

Metabolism of volatile chlorinated aliphatic hydrocarbons by Pseudomonas fluorescens.

A Pseudomonas fluorescens strain designated PFL12 was isolated from soil and water that were contaminated with various chloroaliphatic hydrocarbons. The isolate was able to metabolize 1,2-dichloroethane, 1,1,2-trichloroethane, 1,2-dichloropropane, 2,2-dichloropropane, and trichloroethylene.     

A theory of the symmetries of filamentous bacteriophages.

A mathematical model is presented which explains the symmetries observed for the protein coats of filamentous bacterial viruses. Three viruses (Ff, IKe, and If1) all have five-start helices with rotation angles of 36 degrees and axial translations of 16 A (Type I symmetry), and three other viruses (Pf1, Xf, and Pf3) all have one-start helices with rotation angles of approximately equal to 67 degrees and translations of approximately 3 A (Type II symmetry). The coat protein subunits in each group diverge from each other in amino acid sequence, and Type II viruses differ dramatically in DNA structure. Regardless of the differences, both Type I and Type II symmetry can be understood as direct, natural consequences of the close-packing of alpha-helical protein subunits. In our treatment, an alpha-helical subunit is modeled as consisting of two interconnected, flexible tubular segments that follow helical paths around the DNA, one in an inner layer and the other in an outer layer. The mathematical model is a set of algebraic equations describing the disposition of the flexible segments. Solutions are described by newly introduced symmetry indices and other parameters. An exhaustive survey over the range of indices has produced a library of all structures that are geometrically feasible within our modeling scheme. Solutions which correspond in their rotation angles to Type I and Type II viruses occur over large ranges of the parameter space. A few solutions with other symmetries are also allowed, and viruses with these symmetries may exist in nature. One solution to the set of equations, obtained without any recourse to the x-ray data, yields a calculated x-ray diffraction pattern for Pf1 which compares reasonably with experimental patterns. The close-packing geometry we have used helps explain the near constant linear mass density of known filamentous phages. Helicoid, rigid cylinder, and maximum entropy structure models proposed by others for Pf1 are reconciled with the flexible tube models and with one another.     

Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.     

Potassium channel "inactivation" induced by soft-glass patch pipettes

We have studied the potassium currents of rat pituitary pars intermedia cells kept in primary culture using whole-cell recording with patch pipettes. The potassium current recorded with hard-glass pipettes is mainly carried by voltage-dependent channels that show slow inactivation in the presence of 0.5 mM internal EGTA. Fast "inactivation" of the potassium current is seen with patch pipettes fabricated from soft glass (soda glass or potash lead glass), and is probably caused by block of the potassium channels by di- or multivalent cations released from the glass.