Vapor-phase and fine particulate matter concentrations of polycyclic aromatic hydrocarbons measured during the winter months in a northern Rocky Mountain urban airshed.

A fine particulate matter (PM2.5) sampling program was conducted in Missoula, MT, to investigate both the particle and vapor phases of PM2.5-associated polycyclic aromatic hydrocarbons (PAHs) found in a northern Rocky Mountain urban airshed. Twenty-four-hour samples were collected during the cold winter months of January through April 2002, when many of the more volatile organic components of PM2.5 were expected to be found in the condensed particle form. To meet analytical detection limits, each of the 12 individual sample days were aggregated into four total filter and polyurethane foam (PUF) samples, respectively, with each aggregate containing 3 sample days. Quartz filter (particle-phase PAHs) and PUF (vapor-phase PAHs) aggregates were analyzed separately for 18 individual PAHs and phenolics by gas chromatography/mass spectrometry. Results showed that 87% of the PM2.5-associated phenolics and PAHs measured in this study were found in the vapor phase. PM2.5-associated gas/particle partition coefficients (Kp,2.5) ranged from 0 for the lighter phenolics and PAHs to approximately 0.1 for some of the heavier PAHs, such as fluoranthene and pyrene. Calculating Kp,2.5 for the heaviest measured PAHs was not feasible because of low or undetectable concentrations in the vapor phases of these compounds. Phenolics and two-ringed and three-ringed PAHs were found almost exclusively in the vapor phase. Four-ringed PAHs were distributed between the particle and vapor phases, with more mass measured in the vapor phase. Very little five-ringed and higher PAHs were measured from either the filter or PUF sampling medium. These results provide information on both the concentrations and different phases of PM2.5-associated PAHs measured during the winter months in a northern Rocky Mountain urban airshed, when concentrations of PM2.5 are generally at their highest compared with the rest of the year.

The Missoula Valley semivolatile and volatile organic compound study: seasonal average concentrations.

The city of Missoula is located in a high mountain valley (elevation 3200 ft.) in western Montana and contains one of the largest populations in the entire Rocky Mountain Region completely enclosed by mountains. During the 2000/2001 Missoula Valley Sampling Program, ambient levels of 61 semivolatile organic compounds (SVOCs) and 54 volatile organic compounds (VOCs) were originally quantified before refining the analytical program to 28 of the most prominent SVOCs and VOCs found in the Missoula Valley airshed. These compounds were measured over 24-hr periods at two locations throughout an entire year. This study provides the first, comprehensive appraisal of the levels of SVOCs and VOCs measured simultaneously throughout all four seasons at two locations in the Missoula Valley, including those levels measured during the 2000 Montana wildfire season. Generally, SVOC levels were comparable between both sides of the Missoula Valley. However, there were nearly double the amount of VOCs measured at the more urban Boyd Park site compared with the rural Frenchtown sampling site, a result of the greater number of automobiles on the eastern side of the Valley. SVOCs and VOCs were measured at their highest levels of the sampling program during the winter. Forest fire smoke samples collected during the summer of 2000 showed significant increases in SVOC phenolic compounds, including phenol, 2-methylphenol, 4-methylphenol, and 2,4-dimethylphenol. Although there were modest increases in some of the other SVOCs and VOCs measured during the fire season, none of the increases were as dramatic as the phenolics.

Decreased uptake of bodipy-labelled compounds in the presence of the nuclear stain, DRAQ5.

We have found the nuclear stain DRAQ5 to decrease the cellular uptake of a series of boron dipyrromethane (bodipy)-labelled compounds. This phenomenon is consistent between Lysotracker Green DND 26, Lysotracker Red DND 99 and bodipy-labelled mycolactone. Although DRAQ5 uptake was not prevented, DRAQ5 was in significant excess in each case. As the effect is consistent among two cell types, RAW 264.7 monocyte/macrophages and Bend 3 endothelial cells, we hypothesize that it may be a result of the two dyes complexing in solution into a form that is not taken up by the cells. This hypothesis is confirmed by fluorescence resonance energy transfer analysis in solution.

Identification of 11 pH-regulated genes in Borrelia burgdorferi localizing to linear plasmids.

When Borrelia burgdorferi is transmitted from the tick vector to the mammalian host, the bacterium experiences alterations in its environment, such as changes in temperature and pH. Previously, we observed numerous alterations in the membrane protein profile when B. burgdorferi B31 was grown at pH 7.0 compared to pH 8.0. Here we identify 11 genes localizing to linear plasmids that are up-regulated at pH 7.0 relative to pH 8.0 in vitro. Seven genes (bba03, bba24, bba64, bba66, bbe31, bbj41/bbi39 [encoding products that are 99% identical], and bbk01) were indirectly identified by proteomic analysis of membrane proteins. Another gene, bba36, was identified by screening a B. burgdorferi B31 genomic library with cross-adsorbed hyperimmune rabbit serum. Two additional genes, bba65 and bba73, were identified by Northern blot analysis. Genes bba64, bba65, bba66, bbj41/bbi39, and bba73 are members of paralogous gene family 54, and bbe31 is a member of the closely related paralogous gene family 60. Gene bba24 is part of a bicistronic operon with bba25 that encodes the well-characterized decorin binding proteins A and B. All 11 genes were transcriptionally regulated, yet the degree of pH regulation varied, with some genes more tightly regulated than others. The regions upstream of these pH-regulated genes appeared to be unrelated, yet many contained dyad repeats ranging from 12 to 25 nucleotides in length that may be involved in the regulation of these genes.

Bacteriophages of spirochetes.

Historically, a number of bacteriophage-like particles have been observed in association with members of the bacterial order Spirochetales, the spirochetes. In the last decade, several spirochete bacteriophages have been isolated and characterized at the molecular level. We have recently characterized a bacteriophage of the Lyme disease agent, Borrelia burgdorferi, which we have designated phiBB-1. Here we review the history of the association between the spirochetes and their bacteriophages, with a particular emphasis on phiBB-1 and its prophage, the 32-kb circular plasmid family of B. burgdorferi.

A tubular network associated with the brush-border surface of the Aedes aegypti midgut: implications for pathogen transmission by mosquitoes.

The mosquito Aedes aegypti is capable of transmitting a variety of pathogens to man and to other vertebrates. The midgut of this insect has been well-studied both as the tissue where the first contact occurs between ingested pathogens and the insect host, and as a model system for blood meal digestion in blood-sucking insects. To understand better the nature of the midgut surface encountered by parasites or viruses, we used scanning electron microscopy to identify the most prominent structures and cell morphologies on the luminal midgut surface. The luminal side of the midgut is a complex and layered set of structures. The microvilli that are found on most, but not all, cells are covered by a network of fine strands that we have termed the microvilli-associated network (MN). The MN strands are membranous, as shown by a membrane bilayer visible in cross sections of MN strands at high magnification in transmission electron micrographs. The MN is found in blood-fed as well as unfed mosquitoes and is not affected by chitinase treatment, suggesting that it is not related to the chitinous peritrophic membrane that is formed only after blood feeding. The cells in the midgut epithelium have two distinct morphologies: the predominant cell type is densely covered with microvilli, while cells with fewer microvilli are found interspersed throughout the midgut. We used lectins to probe for the presence of carbohydrates on the midgut surface. A large number of lectins bind to the luminal midgut surface, suggesting that a variety of sugar linkages are present on the structures visualized by electron microscopy. Some of these lectins partially block attachment of malaria ookinetes to the midgut surface in vitro. Thus, the mosquito midgut epithelium, like the lining of mammalian intestines, is complex, composed of a variety of cell types and extensively covered with surface carbohydrate that may play a role in pathogen attachment.

Persistence of hepatitis C virus in a human megakaryoblastic leukaemia cell line.

Thrombocytopenia is a frequent clinical finding in patients with hepatitis C virus (HCV) infection. Platelets from patients with HCV infection have been identified as carriers of HCV RNA in our previous studies. The present study was designed to further investigate the possibility of HCV replication in megakaryoblasts from which platelets are eventually released. A megakaryoblastic cell line (MEG-01), established from a chronic myelogenous leukaemia patient 13 years ago, was used for this study. The MEG-01 cells were inoculated with fresh serum from a patient with HCV infection and renamed MEG-01-I cells. Surprisingly, both MEG-01 and MEG-01-I were positive by HCV reverse transcription-polymerase chain reaction (RT-PCR) for the existence of HCV RNA and minus-strand HCV RNA, regardless of inoculation. This was further confirmed by in situ RT-PCR. The HCV antigens, such as core, envelope, and non-structural (NS)3 and NS4, were also present in both cell lines, as identified by Western blotting and indirect immunofluorescence staining. In addition, virus-like particles were observed by electron microscopy in the MEG-01 cell line as well as in the MEG-01-I cell line. These findings indicate that the megakaryoblasts are vulnerable to HCV infection and that replication of HCV can occur in these cells. This may help us to better understand the pathogenesis of thrombocytopenia in patients with HCV infection. The MEG-01 cell line, which may have been continuously shedding HCV for years, should be a useful model for experimental research into HCV.

Idiopathic thrombocytopenic purpura in a cat.

An 11-year-old, castrated, male domestic shorthair cat was presented for hematuria and pollakiuria. The cat had a marked thrombocytopenia, and a bone-marrow core biopsy demonstrated megakaryocytic hyperplasia with many megakaryocyte-associated neutrophils (i.e., emperipolesis). On peripheral blood, collected at initial presentation, what appeared to be platelets were noted to be within or adherent to occasional neutrophils. The thrombocytopenia was idiopathic in that no definitive cause could be found. However, platelet concentrations appeared to increase and decrease in response to changes in prednisone and cyclosporine therapy, suggesting a possible immune-mediated pathogenesis. As tests to detect increased feline platelet-associated antibodies are unavailable, immune-mediated thrombocytopenia can only be tentatively diagnosed in cats by exclusion and response to therapy.

Effects of environmental pH on membrane proteins in Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, alternates between the microenvironments of the tick vector, Ixodes scapularis, and a mammalian host. The environmental conditions the spirochete encounters during its infectious cycle are suspected to differ greatly in many aspects, including available nutrients, temperature, and pH. Here we identify alterations in the membrane protein profile, as determined by immunoblotting and two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), that occur in virulent B. burgdorferi B31 as the pH of the medium is altered. Initial comparisons of cultures incubated at pHs 6.0, 7.0, and 8.0 yielded alterations in the expression of seven membrane proteins as determined by probing with hyperimmune rabbit serum. Six of these membrane proteins (54, 45, 44, 43, 35, and 24 kDa) were either present in increased amounts in or solely expressed by cultures incubated at pHs 6.0 and 7.0. The 24-kDa protein that decreased in expression at pH 8.0 was identified as outer surface protein C (OspC). In addition, a 42-kDa membrane protein increased in amount in cultures incubated at pH 8.0. Similar changes were observed with serum from a mouse infected by tick bite, with the recognition of two additional bands (48 and 46 kDa) unique to pHs 6. 0 and 7.0. When membrane fractions were analyzed by 2D-NEPHGE, at least 37 changes in the membrane protein profile between cells incubated at pHs 6.0, 7.0, and 8.0 were observed by immunoblotting and silver staining. Environmental cues such as pH may prove important in the regulation of virulence determinants and factors necessary for the adaptation of B. burgdorferi to the tick or mammalian microcosm.

A cryptic 19-kilobase plasmid associated with U.S. isolates of Yersinia pestis: a dimer of the 9.5-kilobase plasmid.

Yersinia pestis, the etiologic agent of plague, carries three prototypic plasmids with sizes of 110 kb (pFra, pTox), 70 kb (pLcr, pVW, pCad), and 9.5 kb (pPla, pPst). Studies suggest that geographic isolates of Y. pestis may be differentiated by plasmid profiles. Yersinia pestis isolated from the western United States harbor an additional plasmid, estimated to be approximately 19 kb in size. This cryptic plasmid was characterized by restriction endonuclease digestion, amplification and sequencing of the plasminogen activator gene segment, Southern blotting, and visualized by electron microscopy. Results revealed that this cryptic plasmid is a supercoiled DNA plasmid, 18.85+/-0.59 (mean+/-SD) kb in length, and is a dimer of the 9.5-kb plasmid. The genetic reason for the appearance of this form of the 9.5-kb plasmid in Y. pestis from Arizona, California, Colorado, New Mexico, and Texas is under study.

Adhesion of Plasmodium gallinaceum ookinetes to the Aedes aegypti midgut: sites of parasite attachment and morphological changes in the ookinete.

Plasmodium gallinaceum ookinetes adhered to Aedes aegypti midgut epithelia when purified ookinetes and isolated midguts were combined in vitro. Ookinetes preferentially bound to the microvillated luminal surface of the midgut, and they seemed to interact with three types of structures on the midgut surface. First, they adhered to and migrated through a network-like matrix, which we have termed microvilli-associated network, that covers the surface of the microvilli. This network forms on the luminal midgut surface in response to blood or protein meals. Second, the ookinetes bound directly to the microvilli on the surface of the midgut and were occasionally found immersed in the thick microvillar layer. Third, the ookinetes associated with accumulations of vesicular structures found interspersed between the microvillated cells of the midgut. The origin of these vesicular structures is unknown, but they correlated with the surface of midgut cells invaded by ookinetes as observed by TEM. After binding to the midgut, ookinetes underwent extensive morphological changes: they frequently developed one or more annular constrictions, and their surface roughened considerably, suggesting that midgut components remain bound to the parasite surface. Our observations suggest that, in a natural infection, the ookinete interacts in a sequential manner with specific components of the midgut surface. Initial binding to the midgut surface may activate the ookinete and cause morphological changes in preparation for invasion of the midgut cells.

The antimicrobial agent melittin exhibits powerful in vitro inhibitory effects on the Lyme disease spirochete.

Borrelia burgdorferi has demonstrated a capacity to resist the in vitro effects of powerful eukaryotic and prokaryotic metabolic inhibitors. However, treatment of laboratory cultures on Barbour-Stoenner-Kelly medium with melittin, a 26-amino acid peptide contained in honeybee venom, showed immediate and profound inhibitory effects when they were monitored by dark-field microscopy, field emission scanning electron microscopy, and optical density measurements. Furthermore, at melittin concentrations as low as 100 microg/mL, virtually all spirochete motility ceased within seconds of inhibitor addition. Ultrastructural examination of these spirochetes by scanning electron microscopy revealed obvious alterations in the surface envelope of the spirochetes. The extraordinary sensitivity of B. burgdorferi to mellitin may provide both a research reagent useful in the study of selective permeability in microorganisms and important clues to the development of effective new drugs against lyme disease.

Cloning and expression of the Borrelia burgdorferi lon gene.

The ATP-dependent protease Lon (La) of Escherichia coli degrades abnormal proteins and is involved in the regulation of capsular polysaccharide synthesis. In addition, mutations in the E. coli lon gene suppress temperature-sensitive mutations in other genes. The lon gene of Borrelia burgdorferi, encoding a homolog of the Lon protease, has been cloned and sequenced. The gene encodes a protein of 806 amino acids. The deduced amino acid sequence of the B. burgdorferi Lon protease shares substantial sequence identity with those of other known Lon proteases. The transcription start point of the B. burgdorferi lon gene was identified by primer extension analysis and the potential promoter did not show similarities to the consensus heat-shock promoter in E. coli. The 5'-end of the B. burgdorferi lon gene appears to suppress the temperature-sensitive phenotype of an E. coli lpxA mutant.

Polysialic acid export in Escherichia coli K1: the role of KpsT, the ATP-binding component of an ABC transporter, in chain translocation.

The polysialic acid (polySia) capsule of Escherichia coli K1 is a key virulence determinant of the organism, allowing it to evade host defenses. The proteins necessary for expression of the capsule are encoded by the 17 kb kps gene cluster. This cluster contains two genes, kpsM and kpsT, that are required for polySia transport across the cytoplasmic membrane. KpsM is a hydrophobic integral inner membrane protein, while KpsT is a peripheral inner membrane protein that binds ATP. They belong to the ATP-binding cassette (ABC) superfamily of transporters. To study the role of KpsT in polySia translocation, we used PCR mutagenesis to isolate dominant negative mutations of plasmid-encoded kpsT. All mutations mapped to the same glutamic acid residue at position 150, adjacent to Walker motif B of KpsT. Wild-type (kps+) cells harboring one such allele, E150G, did not transport polySia to the cell surface but accumulated intracellular polysaccharide and produced small colonies containing cells that grew as long filaments. The E150G protein still bound ATP as shown by 8-azidoATP photolabeling assays. We combined the E150G allele with each of five mutations isolated previously in kpsT. Mutations that disrupt ATP-binding (K44E) or alter regions of the protein thought to interact with KpsM (G84D, S126F) suppressed the dominant negative phenotype while mutations in the C-terminal portion of the protein (C163Y, H181Y) did not suppress. These studies have allowed the development of a working model for the role of KpsT in polySia chain translocation.

Variation in nectar volume and composition of Impatiens capensis at the individual, plant, and population levels.

Although the volume and chemical composition of nectars are known to vary among plant species and to affect pollinator response to plants, relatively little is known of the variation in volume, and sugar and amino acid composition within species. We collected nectar from Impatiens capensis in a nested design: three flowers from each of three plants from each of three populations. This design enabled us to quantify variation within individual plants, among plants within populations, and among populations. Using high performance liquid chromatography, we analyzed the sugar and amino composition of the 27 flowers. Analysis of variance showed that none of the parameters (volume, concentrations of three sugars and 24 amino compounds) varied within individuals. Variation in nectar volume was not significant among plants but was nearly significant among populations. Of the three sugars detected (sucrose, glucose, and fructose), the only significant variation was that of sucrose among populations. Concentrations of 12 amino compounds varied significantly at the plant level while 7 amino compounds varied among populations. The results indicate that: (1) pooling of nectar samples from flowers of individual plants can be an acceptable methodology for those seeking to understand within species variation; (2) amino compounds appear to vary more than either volumes or sugar concentrations; (3) future studies should assess how much of the observed variation is due to genetic versus environmental differences; (4) additional studies should examine the geographic variation in nectar parameters and pollinators of I. capensis in order to assess the role different pollinators play in shaping nectar composition.

Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB.

No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the physiology and pathogenesis of the Lyme disease agent on a molecular genetic level.

Analysis of the distribution and molecular heterogeneity of the ospD gene among the Lyme disease spirochetes: evidence for lateral gene exchange.

Analysis of the ospD gene has revealed that this gene is not universal among Lyme disease spirochete isolates. The gene was found to be carried by 90, 50, and 24% of the Borrelia garinii, B. afzelii, and B. burgdorferi isolates tested. Size variability in the ospD-encoding plasmid was also observed. Sequence analysis has demonstrated the presence of various numbers of a 17-bp repeated sequence in the upstream control (promoter) region of the gene. In addition, a region within the coding sequence where various insertions, deletions, and direct repeats occur was identified. ospD gene sequences from 31 different isolates were determined and utilized in pairwise sequence comparisons and construction of a gene tree. These analyses suggest that the ospD gene was the target of several recombinational events and that the gene was recently acquired by Lyme disease spirochetes and laterally transferred between species.

gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi.

We have isolated and characterized mutants of Borrelia burgdorferi that are resistant to the antibiotic coumermycin A1, which targets the B subunit of DNA gyrase. Mutants had either 100- or 300-fold higher resistance to coumermycin A1 than wild-type B. burgdorferi. In each case, a single point mutation in the gyrB gene converted Arg-133 to Gly or Ile. Mutations in the homologous Arg residue of Escherichia coli DNA gyrase are also associated with resistance to coumarin antimicrobial agents.

Variation in the size of the ospA-containing linear plasmid, but not the linear chromosome, among the three Borrelia species associated with Lyme disease.

The aetiological agents of Lyme disease form a phylogenetically heterogeneous group, composed of three species, Borrelia burgdorferi, Borrelia garinii, and group VS461. We have compared the sizes of the linear plasmid that carries the genes encoding the major outer-surface proteins OspA and OspB as well as the size and structure of the chromosome among the Lyme disease spirochaetes. We have found differences in the sizes of the ospA-containing plasmids, but not the linear chromosomes among the three species. The ospA-containing plasmid size of 50 kb in B. burgdorferi isolates is significantly smaller than the size of 55 kb in B. garinii isolates and 56 kb in group VS461 isolates. The chromosome was found to be linear in all three Borrelia species, but not significantly different in size.