Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting.

The use of fluorescence-activated flow cytometric cell sorting to obtain highly enriched populations of viable target bacteria was investigated. Preliminary studies employed mixtures of Staphylococcus aureus and Escherichia coli. Cells of S. aureus, when mixed in different proportions with E. coli, could be selectively recovered at a purity in excess of 90%. This was possible even when S. aureus composed only approximately 0.4% of the total cells. Cell sorting was also tested for the ability to recover E. coli from natural lake water populations and sewage. The environmental samples were challenged with fluorescently labelled antibodies specific for E. coli prior to cell sorting. Final sample purities of greater than 70% were routinely achieved, as determined by CFU. Populations of E. coli released into environmental samples were recovered at greater than 90% purity. The use of flow cytometry and cell sorting to detect and recover viable target bacteria present at levels of less than 1% within an indigenous microflora was also demonstrated.     

Encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type 1 Gag and Pol proteins upon infection.

The use of recombinant viruses for the expression of a wide array of foreign proteins has become commonplace during the last few years. Recently, we have described the construction and characterization of chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes in which the gag and pol genes of HIV-1 have been substituted for the VP2 and VP3 capsid genes of the P1 capsid precursor region of poliovirus. Transfection of these RNAs into tissue culture cells results in replication of the RNA genome and expression of HIV-1-P1 fusion proteins (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Here we report on the encapsidation and amplification of the minireplicons to obtain sufficient quantities for biological characterization. To do this, HIV-1-poliovirus minireplicon genomes containing the gag or pol gene were transfected into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid precursor protein, P1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). The chimeric minireplicons replicated and expressed the appropriate HIV-1-P1 fusion proteins as determined by immunoprecipitation with HIV-1-specific antibodies. The encapsidated genomes were isolated by ultracentrifugation. Reinfection of cells with the encapsidated chimeric RNA genomes resulted in expression of the HIV-1-Gag-P1 or HIV-1-Pol-P1 fusion protein. Serial passaging of the encapsidated chimeric HIV-1-poliovirus genomes was accomplished by coinfecting cells with the encapsidated minireplicons and VV-P1, resulting in stocks of the encapsidated minireplicons. Northern (RNA) blot analysis of passaged material revealed that no detectable deletions of the chimeric genomes occurred during 14 serial passages. Infection of cells by the encapsidated minireplicons was blocked by antipoliovirus antibodies. Coinfection of cells with encapsidated minireplicons and type 1 Sabin poliovirus resulted in encapsidation of the chimeric genomes by wild-type poliovirus as measured by immunoprecipitation of the HIV-1-P1 fusion proteins with HIV-1-specific antibodies. The results of this study demonstrate the encapsidation of poliovirus minireplicons which express foreign proteins and point to the future use of this system as a potential vaccine vector.     

Evolutionarily Conserved, “Acatalytic” Carbonic Anhydrase-Related Protein XI Contains a Sequence Motif Present in the Neuropeptide Sauvagine: The Human CA-RP XI Gene (CA11) Is Embedded between theSecretorGene Cluster and theDBPGene at 19q13.3

Conserved amino acid motifs are found in numerous expressed genes. Proteins and peptides with functional relationships may be identified using probes designed to hybridize with these motifs. An oligonucleotide probe was prepared to match the sequence of the expected active region of a frog corticotropin-releasing factor-like peptide sauvagine and used to screen a sheep brain cDNA library. A novel 1331-bp cDNA encoding a putative 328-residue protein with a theoretical mass of 36 kDa was identified. The presence of a strong signal sequence indicates that it is a secreted protein. The amino- and carboxy-terminal regions are characterized by several potential phosphorylation sites and binding motifs, suggesting a role in intracellular signal transduction. Although the protein possesses a 7-residue sequence identical to that found in sauvagine, its overall primary structure most closely resembles those of the α-carbonic anhydrases (α-CAs). Moreover, the detection of the human and mouse orthologues in the EST databases, together with an evolutionary analysis, indicates that the protein represents a new member of the α-CA gene family, which we designate carbonic anhydrase-related protein XI (CA-RP XI), encoded byCA11(human) andCar11(mouse, rat). The humanCA11gene appears to be located between the secretor type α(1,2)-fucosyltransferase gene cluster (FUT1–FUT2–FUT2P) and the D-site binding protein gene (DBP) on chromosome 19q13.3. Despite potentially inactivating changes in the active-site residues, CA-RP XI is evolving very slowly in mammals, a property indicative of an important function, which has also been observed in the two other “acatalytic” CA isoforms, CA-RP VIII and CA-RP X, whose functions are unknown.     

A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation.     

An evaluation of lectin-mediated magnetic bead cell sorting for the targeted separation of enteric bacteria

Lectin-labelled magnetic beads were assessed and compared with antisera as an alternative approach for the targeted separation and isolation of enteric bacteria. Of the 16 lectins tested against a range of bacterial species, concanavalin A (conA) showed the greatest potential. Agglutination of bacterial cells in suspension using conA and methods for effective labelling of the magnetic beads with the lectin were optimized. ConA-labelled magnetic beads were compared with antibody-labelled beads for recovery of bacterial cells from pure or mixed laboratory cultures and from natural populations in river water. Recovered cell populations were free from environmental impurities and a high percentage of the culturable cells was extracted. Specific cell recovery was found to be variable, but the use of lectins offers some promise as an alternative cell discriminator.     

Site-Specific Prebiotic Oligomerization Reactions of Glycine on the Surface of Hectorite

Condensation reactions of the amino acid glycine on the surface of Cu(II)-exchanged hectorite are investigated using the technique of scanning force microscopy. Prebiotic conditions are simulated using alternate wetting and heating cycles. Concentration, immobilization, and subsequent polymerization resulting in glycine oligomers are seen to occur primarily at step edges or faults in the topmost layer. Condensation reactions also occur within tiny micropores or defects in the topmost layer. These reactions are facilitated by the availability of intergallery metal cations at the step edges or pores in the surface region. Received: 19 January 1998 / Accepted: 24 April 1998     

Population genetic variation in rare and endangered Iliamna (Malvaceae) in Virginia

Random amplified polymorphic DNA (RAPD) markers were used as input for an analysis of molecular variance (AMOVA), homogeneity of molecular variance analysis (HOMOVA), and cluster analysis to describe the population genetic structure of Iliamna corei, a federally endangered plant located only in Virginia, and I. remota , a rare plant in Virginia, Indiana, and Illinois. The analysis was performed to help clarify the taxonomic relationship between the two closely related species. We analysed four clones in the only known population of I. corei , breeding stock derived from seeds originating from the population site, and three I. remota populations in Virginia. Eighty-five percent of screened primers revealed DNA polymorphisms in Iliamna. Ninety-nine informative markers were generated using seven primers. No significant statistical differences (at P = 0.05) in RAPD variation was found between species (24% of variance) using the AMOVA procedure. However, within species/among populations (31 % of the variance) and within populations (45% of the variance) there were significant differences (P < 0.002). An unweighted paired group method using arithmetic averages (UPGMA) cluster analysis showed the federally endangered I. corei population to be genetically distinct from the apparently recently introduced (in Virginia: ∼ 100 ybp) I. remota. The lack of significant differences from the AMOVA and the high number shared bands between I. corei and I. remota suggest that I. corei may be more appropriately classified as a subspecies of I. remota. Iliamna corei plants in the natural population were genetically similar to one another while the I. corei breeding stock plants and I. remota plants were genetically relatively diverse.     

Characterization of the ATP-sensitive binding of Tetrahymena 30 S dynein to bovine brain microtubules

Dynein was obtained by high salt extraction of Tetrahymena cilia and purified by DEAE-Sephacel chromatography. This fraction consisted of a mixture of 30 S dynein (80%) and the 14 S ATPase (15%). The column purification effectively removed tubulin and adenylate kinase. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the 30 S dynein was composed of a major heavy chain (approximately 400 kD, three copies), three intermediate chains (70, 85, and 100 kD), and a group of light chains (approximately 20 kD). The binding of the column-purified dynein to bovine brain microtubules was characterized as follows. (i) Titration of the dynein with microtubules showed a linear increase in turbidity up to an equivalence point of 2.7 mg of dynein/mg of tubulin with apparently tight binding; (ii) the addition of ATP caused the turbidity of the solution of decrease to a level equal to the sum of free dynein plus microtubules; (iii) transmission electron microscopy indicated that microtubules were decorated with dynein arms spaced at a 24-nm longitudinal repeat and that the dynein decoration was removed upon addition of ATP; (iv) cross-section images of microtubules that were saturated with dynein showed six to seven dynein arms around a microtubule consisting of 14 protofilaments, corresponding to a molar ratio of one dynein/six tubulin dimers; (v) the dynein arms were bound primarily by their broader end which corresponds to the end normally bound to the B-subfiber in vivo. Experiments with purified 30 and 14 S dyneins indicated that the dynein-microtubule binding activity and the ATP-induced dissociation were the properties of the 30 S dynein alone. These studies demonstrate that the 30 S dynein under our conditions (50 mM PIPES, pH 6.96, 4 mM MgSO4) interacts with bovine brain microtubules through the ATP-sensitive site of the dynein arm.     

Regulation of bacterial glycogen synthesis. Stimulation of glycogen synthesis by endogenous and exogenous cyclic adenosine 3':5'-monophosphate in Escherichia coli and the requirement for a functional CRP gene

In Escherichia coli cya mutants, deficient in adenylate cyclase (EC 4.6.1.1), basal cellular rates of glycogen synthesis were lower and the relative increases produced by exogenous cyclic adenosine 3',5'-monophosphate during growth on glucose were greater than in their respective parent strains. These observations provide strong evidence that endogenous cyclic AMP is one of the key regulators of glycogen synthesis in growing E. coli. In crp mutants, deficient in cyclic AMP receptor protein (CRP), the basal cellular rates of glycogen synthesis were much lower than in their respective parent strains. Stimulation of glycogen synthesis by exogenous cyclic AMP was markedly attenuated in the three crp mutants. Thus, stimulation of glycogen synthesis by either endogenous or exogenous cyclic AMP appears to require CRP. Functional CRP appeared to be required for all three responses observed after cyclic AMP addition: an abrupt step-up in the cellular rate of glycogen synthesis, a continuing exponential increase in rate, and a stimulation of the rate during a subsequent nitrogen starvation. To account for these responses, we derived a mathematical model in which the cyclic AMP-CRP complex regulates the differential rate of synthesis of an enzyme metabolizing an effector of the rate-limiting enzyme of glycogen synthesis.