Characterization of Bacteria by Particle Beam Mass Spectrometry

A technique is described for detecting and characterizing bacteria on a single-particle basis by mass spectrometry. The method involves generation of a particle beam of single whole cells which are rapidly volatilized and ionized in vacuum in the ion source of a quadrupole mass spectrometer. The particle beam can be generated, with minimal sample handling, from a naturally occurring aerosol or from a solution of bacteria that can be dispersed as an aerosol. The mass spectrum is generated by successively measuring the average intensities of different mass peaks. The average intensity is obtained by measuring the ion intensity distribution at the particular mass (m/e) for ion pulses from more than 1,000 bacteria particles. Bacillus cereus, Bacillus subtilis, and Pseudomonas putida samples were analyzed to test the capability of the instrument for differentiating among species of bacteria. Significant ion-intensity information was produced over the m/e range of 50 to 300, an improvement over previous pyrolysis-mass spectrometry results. The complex mass spectra contained a few unique peaks which could be used for the differentiation of the bacteria. A statistical analysis of the variations in peak intensities among the three bacteria provided a quantitative measure of the reproducibility of the instrument and its ability to differentiate among bacteria. The technique could lead to a new rapid method for the analysis of microorganisms and could be used for the detection of airborne pathogens on a continuous, real-time basis.     

Conductimetric assessment of the biomass content in suspensions of immobilised (gel-entrapped) microorganisms

Summary The problem of obtaining a rapid estimate of the microbial content of an immobilised cell suspension is addressed. The low-frequency conductivity of free-living cell suspensions of Clostridium pasteurianum is lower than that of the medium in which they are suspended, by an amount conforming to the Bruggeman relation. The conductivity of the cell wall makes a negligible contribution to the measured conductivity under the conditions used. Calcium alginate beads (lacking microbial cells) lower the conductivity of a solution with which they have been equilibrated by an extent which is a function of the concentration of alginate gel used in forming the beads. When this is taken into account, the ratio of the conductivity of a suspension of gel-immobilised cells to that of the suspending medium can be used to give a rapid and convenient assessment of the amount of microbial biomass present.     

The Diffusion of Oxygen, Carbon Dioxide, and Inert Gas in Flowing Blood

Measurements were made of exchange rates of oxygen, carbon dioxide, and krypton-85 with blood at 37.5°C. Gas transfer took place across a 1 mil silicone rubber membrane. The blood was in a rotating disk boundary layer flow, and the controlling resistance to transfer was the concentration boundary layer. Measured rates were compared with rates predicted from the equation of convective diffusion using velocities derived from the Navier-Stokes equations and diffusivities calculated from the theory for conduction in a heterogeneous medium. The measured absorption rate of krypton-85 was closely predicted by this model. Significant deposition of material onto the membrane surface, resulting in an increased transfer resistance, occurred in one experiment with blood previously used in a nonmembrane type artificial lung. The desorption rate of oxygen from blood at low P_o2¹ was up to four times the corresponding transfer rate of inert gas. This effect is described somewhat conservatively by a local equilibrium form of the convective diffusion equation. The carbon dioxide transfer rate in blood near venous conditions was about twice that of inert gas, a rate significantly greater than predicted by the local equilibrium theory. It should be possible to apply these theoretical methods to predict exchange rates with blood flowing in systems of other geometries.     

Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells

Anthrax lethal toxin consists of two separate proteins, protective antigen and lethal factor (LF). Certain macrophages and a mouse macrophage-like cell line, J774A.1, are lysed by low concentrations of lethal toxin. In contrast, another macrophage cell line, IC-21, and all other cell types tested were resistant to this toxin. To discover the basis for this difference, each step in the intoxication process was examined. No differences between sensitive and resistant cells were found in receptor binding or proteolytic activation of protective antigen, steps that are required prior to LF binding. To determine whether resistance results from a defect in translocation to the cytosol, we introduced LF into J774A.1 and IC-21 cells and a nonmacrophage cell line (L6 myoblast) by osmotic lysis of pinocytic vesicles. Only J774A.1 cells were lysed; no effect was observed in IC-21 and L6 cells. These results suggest that resistant cells either lack the intracellular target of LF or fail to process LF to an active form. The relatively low potency of LF introduced into J774A.1 cells by osmotic lysis suggests that protective antigen may also be required at a stage subsequent to endocytosis.     

Cold responses of Arabidopsis mutants impaired in freezing tolerance

Mutants of Arabidopsis thaliana L. (Heynh), characterized asdeficient in their freezing tolerance after cold acclimation,were surveyed for some of the normal responses to cold exposure.In foliar tissue, the coldinducibility of three proteins, thelevels of sucrose and glucose, the fatty acyl composition oflipids, and the accumulation of anthocyanin was examined. Fourmutations (sfr3, sfr4, sfr6, and sfr7) reduced or eliminatedthe accumulation of anthocyanin during cold acclimation. Onemutation (sfr4) prevented the normally cold-induced elevationof sucrose and glucose levels; both sfr4 and another mutation(sfr7) affected fatty acid composition after (and only after)cold acclimation. On the other hand mutations sfr1, sfr2 andsfr5 did not differ significantly from the wild type in anyof the parameters tested, suggesting that they have other, perhapshighly specific, effects on lowtemperature responses. Key words: Arabidopsis thaliana, cold acclimation, freezing tolerance, mutation     

Familial Site-specific Ovarian Cancer Is Linked to BRCA1 on 17q12-21

In a study of nine families with “site-specific” ovarian cancer (criterion: three or more cases of epithelial ovarian cancer and no cases of breast cancer diagnosed at age <50 years) we have obtained evidence of linkage to the breast-ovarian cancer susceptibility gene, BRCA1 on 17q12-21. If the risk of cancer in these families is assumed to be restricted to the ovary, the best estimate of the proportion of families linked to BRCA1 is .78 (95% confidence interval .32–1.0). If predisposition to both breast and ovarian cancer is assumed, the proportion linked is 1.0 (95% confidence interval .46–1.0). The linkage of familial site-specific ovarian cancer to BRCA1 indicates the possibility of predictive testing in such families; however, this is only appropriate in families where the evidence for linkage to BRCA1 is conclusive.     

DNA binding of a spermine derivative: Spectroscopic study of anthracene-9-carbonyl-n1-spermine with poly[d(G-C)·(d(G-C))] and poly[d(A-T) · d(A-T)]

The binding of polyamines, including spermidine ( 1 ) and spermine ( 2 ), to poly[d(G-C) · d(G-C) ] was probed using spectroscopic studies of anthracene-9-carbonyl-N¹-spermine ( 3 ); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly [(dG-dC) · (dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C) · d(G-C)] has a site size of 6 ± 1 bases, and an equilibrium binding constant of (2.2 ± 1.1) × 10⁷ M^?1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly [d(A-T) · d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 ± 1.1 bases and an equilibrium binding constant of (6.6 ± 3.3) × 10⁵ M^?1. Thus, 3 binds preferentially to poly [d(G-C) · d(G-C)] at these concentrations. © 1994 John Wiley & Sons, Inc.     

Restriction enzymes in cells, not eppendorfs

Restriction enzymes are essential reagents to molecular biologists, but their relevance to bacterial populations is less obvious. Most bacteria encode restriction and modification systems and these are commonly considered to be a barrier to phage infection. Current evidence also supports a more general role for them in genetic recombination.