bluuebunny/crossref_metadata_2025 huggingface dataset:
Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture
Authors: Meckenstock Rainer U. , Annweiler Eva , Michaelis Walter , Richnow Hans H. , Schink Bernhard
Published: July 2002

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds
ortho
-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3,4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-
13
C]naphthalene or deuterated D
8
-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [
13
C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene,
13
C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.
Influence of Chlorine Substituents on Rates of Oxidation of Chlorinated Biphenyls by the Biphenyl Dioxygenase of
Burkholderia
sp. Strain LB400
Authors: Arnett Clint M. , Parales Juan V. , Haddock John D.
Published: July 2002

Biphenyl dioxygenase from
Burkholderia
(
Pseudomonas
) sp. strain LB400 catalyzes the first reaction of a pathway for the degradation of biphenyl and a broad range of chlorinated biphenyls (CBs). The effect of chlorine substituents on catalysis was determined by measuring the specific activity of the enzyme with biphenyl and 18 congeners. The catalytic oxygenase component was purified and incubated with individual CBs in the presence of electron transport proteins and cofactors that were required for enzyme activity. The rate of depletion of biphenyl from the assay mixture and the rate of formation of
cis
-biphenyl 2,3-dihydrodiol, the oxidation product, were almost equal, indicating that the assay accurately measured enzyme-specific activity. Four classes of CBs were defined based on their oxidation rates. Class I contained 3-CB and 2,5-CB, which gave rates that were approximately twice that of biphenyl. Class II contained 2,5,3′,4′-CB, 2,3,2′,5′-CB, 2,3,4,5-CB, 2,3,2′,3′-CB, 2,4,5,2′,5′-CB, 2,5,3′-CB, 2,5,4′-CB, 2-CB, and 3,4,5-CB, which gave rates that ranged from 97 to 35% of the biphenyl rate. Class III contained only 2,3,4,2′,5′-CB, which gave a rate that was 4% of the biphenyl rate. Class IV contained 2,4,4′-CB, 2,4,2′,4′-CB, 3,4,5,2′-CB, 3,4,5,3′-CB, 3,5,3′,5′-CB, and 3,4,5,2′,5′-CB, which showed no detectable depletion. Rates were not significantly correlated with the aqueous solubilities of the CBs or the number of chlorine substituents on the rings. Oxidation products were detected for all class I, II, and III congeners and were identified as chlorinated
cis
-dihydrodiols for classes I and II. The specificity of biphenyl dioxygenase for the CBs examined in this study was determined by the relative positions of the chlorine substituents on the aromatic rings rather than the number of chlorine substituents on the rings.
Human Adenoviruses and Coliphages in Urban Runoff-Impacted Coastal Waters of Southern California
Authors: Jiang Sunny , Noble Rachel , Chu Weiping
Published: July 2002

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (
r
= 0.32) but was significantly correlated (
r
= 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.
Incidence of Male-KillingRickettsiaspp. (α-Proteobacteria) in the Ten-Spot Ladybird BeetleAdalia decempunctataL. (Coleoptera: Coccinellidae)
Authors: von der Schulenburg J. Hinrich Graf , Habig Michael , Sloggett John J. , Webberley K. Mary , Bertrand Dominique , Hurst Gregory D. D. , Majerus Michael E. N.
Published: July 2002

The diversity of endosymbiotic bacteria that kill male host offspring during embryogenesis and their frequencies in certain groups of host taxa suggest that the evolution of male killing and the subsequent spread of male-killing symbionts are primarily determined by host life history characteristics. We studied the 10-spot ladybird beetle,Adalia decempunctataL. (Coleoptera: Coccinellidae), in which male killing has not been recorded previously, to test this hypothesis, and we also assessed the evolution of the male killer identified by DNA sequence analysis. Our results show thatA. decempunctataharbors male-killingRickettsia(α-proteobacteria). Male-killing bacteria belonging to the genusRickettsiahave previously been reported only for the congeneric two-spot ladybird beetle,Adalia bipunctataL. Phylogenetic analysis ofRickettsiaDNA sequences isolated from different populations of the two host species revealed a single origin of male killing in the genusRickettsia. The data also indicated possible horizontal transfer of symbionts between host species. In addition,A. bipunctatais known to bear at least four different male-killing symbionts in its geographic range two of which coexist in the two locations from whichA. decempunctataspecimens were obtained for the present study. Since only a single male-killing taxon was found inA. decempunctata, we assume that the two closely related ladybird beetle species must differ in the number and/or geographic distribution of male killers. We discuss the importance of these findings to our understanding of the evolution and dynamics of symbiotic associations between male-killing bacteria and their insect hosts.
Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences
Authors: Speksnijder Arjen G. C. L. , Kowalchuk George A. , De Jong Sander , Kline Elizabeth , Stephen John R. , Laanbroek Hendrikus J.
Published: July 2002

A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.
Survival of Salmonellae on and in Tomato Plants from the Time of Inoculation at Flowering and Early Stages of Fruit Development through Fruit Ripening
Authors: Guo Xuan , Chen Jinru , Brackett Robert E. , Beuchat Larry R.
Published: July 2002

The fate of salmonellae applied to tomato plants was investigated. Five
Salmonella
serotypes were used to inoculate tomato plants before and after fruits set, either by injecting stems with inoculum or brushing flowers with it. Ripe tomato fruits were subjected to microbiological analysis. Peptone wash water, homogenates of stem scar tissues, and homogenates of fruit pulp were serially diluted and plated on bismuth sulfite agar before and after enrichment. Presumptive
Salmonella
colonies were confirmed by serological tests, PCR assay using HILA2 primers, and enterobacterial repetitive intergenic consensus PCR. Of 30 tomatoes harvested from inoculated plants, 11 (37%) were positive for
Salmonella
. Of the
Salmonella
-positive tomatoes, 43 and 40%, respectively, were from plants receiving stem inoculation before and after flower set. Two of eight tomatoes produced from inoculated flowers contained
Salmonella
. Higher percentages of surface (82%) and stem scar tissue (73%) samples, compared to pulp of
Salmonella
-positive tomatoes (55%), harbored the pathogen. Of the five serotypes in the inoculum, Montevideo was the most persistent, being isolated from tomatoes 49 days after inoculation, and Poona was the most dominant, being present in 5 of 11
Salmonella-
positive tomatoes. Results suggest that
Salmonella
cells survive in or on tomato fruits from the time of inoculation at flowering through fruit ripening. Tomato stems and flowers are possible sites at which
Salmonella
may attach and remain viable during fruit development, thus serving as routes or reservoirs for contaminating ripened fruit.
Streptococcus suis
Serotypes Characterized by Analysis of Chaperonin 60 Gene Sequences
Authors: Brousseau Ronald , Hill Janet E. , Préfontaine Gabrielle , Goh Swee-Han , Harel Josée , Hemmingsen Sean M.
Published: July 2002

Streptococcus suis
is an important pathogen of swine which occasionally infects humans as well. There are 35 serotypes known for this organism, and it would be desirable to develop rapid methods methods to identify and differentiate the strains of this species. To that effect, partial chaperonin 60 gene sequences were determined for the 35 serotype reference strains of
S. suis
. Analysis of a pairwise distance matrix showed that the distances ranged from 0 to 0.275 when values were calculated by the maximum-likelihood method. For five of the strains the distances from serotype 1 were greater than 0.1, and for two of these strains the distances were were more than 0.25, suggesting that they belong to a different species. Most of the nucleotide differences were silent; alignment of protein sequences showed that there were only 11 distinct sequences for the 35 strains under study. The chaperonin 60 gene phylogenetic tree was similar to the previously published tree based on 16S rRNA sequences, and it was also observed that strains with identical chaperonin 60 gene sequences tended to have identical 16S rRNA sequences. The chaperonin 60 gene sequences provided a higher level of discrimination between serotypes than the 16S RNA sequences provided and could form the basis for a diagnostic protocol.
Characterization of Two New Glycosyl Hydrolases from the Lactic Acid Bacterium
Carnobacterium piscicola
Strain BA
Authors: Coombs Jonna , Brenchley Jean E.
Published: July 2002

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate,
Carnobacterium piscicola
strain BA. A 2.2-kb region corresponding to an α-galactosidase gene,
agaA
, was followed by two genes in the same orientation,
bgaB
, encoding a 2-kb β-galactosidase, and
bgaC
, encoding a structurally distinct 1.76-kb β-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including
Lactococcus lactis
, for which the genome sequence is known. To determine if these sequences encoded enzymes with α- and β-galactosidase activities, we subcloned the genes and examined the enzyme properties. The α-galactosidase, AgaA, hydrolyzes
para
-nitrophenyl-α-
d
-galactopyranoside and has optimal activity at 32 to 37°C. The β-galactosidase, BgaC, has an optimal activity at 40°C and a half-life of 15 min at 45°C. The regulation of these enzymes was tested in
C. piscicola
strain BA and activity on both α- and β-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated β-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two β-galactosidases may act synergistically with the α-galactosidase to degrade other polysaccharides available in the environment.
Benzoate Fermentation by the Anaerobic Bacterium
Syntrophus aciditrophicus
in the Absence of Hydrogen-Using Microorganisms
Authors: Elshahed Mostafa S. , McInerney Michael J.
Published: July 2002

The anaerobic bacterium
Syntrophus aciditrophicus
metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of
S. aciditrophicus
produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of
S
.
aciditrophicus
with the hydrogen-using methanogen
Methanospirillum hungatei
produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the
S. aciditrophicus
pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the
S. aciditrophicus-M. hungatei
coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by
S. aciditrophicus
only in a coculture with a hydrogen user and was not metabolized by
S. aciditrophicus
pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by
S. aciditrophicus
pure cultures until a free energy change (Δ
G
′) of −9.2 kJ/mol was reached (−4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an
S. aciditrophicus
pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by
S. aciditrophicus
pure cultures. These results suggest that benzoate fermentation by
S. aciditrophicus
in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO
2
, and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than −20 kJ/mol, the postulated minimum free energy value for substrate metabolism.
Modification of a Rapid Method for the Identification of Gene-Specific Polymorphisms inCryptosporidiumparvumand Its Application to Clinical and Epidemiological Investigations
Authors: Elwin K. , Chalmers R. M. , Roberts R. , Guy E. C. , Casemore D. P.
Published: July 2002

The application of genotyping to clinical isolates ofCryptosporidiumhas increased significantly our knowledge and understanding of the distribution and epidemiology of this parasite. However, some methods can be laborious and demand specialist technical expertise. PCR-restriction fragment length polymorphism (RFLP) techniques represent a more rapid and simple method of genotyping to support epidemiological and clinical investigations than conventional DNA analytical techniques. We describe a nested PCR-RFLP technique that identifies polymorphisms in theC.parvumthrombospondin-related adhesive protein gene locus; this method offers a sensitive and specific tool for the confirmation and investigation of disease associated withC.parvum. The potential of this enhanced method is demonstrated by its application to the confirmation and epidemiological investigation of an outbreak of cryptosporidiosis associated with a school visit to an open farm.
Synthesis of a Low-Molecular-Weight Form of Exopolysaccharide by
Bradyrhizobium japonicum
USDA 110
Authors: Louch Heather A. , Miller Karen J.
Published: July 2002

A novel extracellular low-molecular-weight polysaccharide was detected as a contaminant within extracellular cyclic β-1,6-β-1,3-glucan preparations from
Bradyrhizobium japonicum
USDA 110 cultures. Compositional analysis, methylation analysis, and nuclear magnetic resonance analysis revealed that this low-molecular-weight polysaccharide was composed of the same pentasaccharide repeating unit previously described for the high-molecular-weight form of the exopolysaccharide (EPS) synthesized by
B. japonicum
strains. Mass spectrometry analysis indicated that the size of this low-molecular-weight form of EPS was consistent with a dimeric form of the pentasaccharide repeating unit.
Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for
Lactococcus lactis
Authors: Émond Éric , Lavallée Richard , Drolet Geneviève , Moineau Sylvain , LaPointe Gisèle
Published: July 2002

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative
cis
-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the
cis
-acting
ori
region and the
repB
gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in
Lactococcus lactis
. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of
L. lactis
DNA and has no selection marker. The companion pCOM1 is a
repB
-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in
L. lactis
if
trans
complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of
L. lactis
and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.
Overproduction of
l
-Lysine from Methanol by
Methylobacillus glycogenes
Derivatives Carrying a Plasmid with a Mutated
dapA
Gene
Authors: Motoyama Hiroaki , Yano Hiroshi , Terasaki Yoko , Anazawa Hideharu
Published: July 2002

The
dapA
gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by
l
-lysine, was cloned from an
l
-threonine- and
l
-lysine-coproducing mutant of the obligate methylotroph
Methylobacillus glycogenes
DHL122 by complementation of the nutritional requirement of an
Escherichia coli dapA
mutant. Introduction of the
dapA
gene into DHL122 and AL119, which is the parent of DHL122 and an
l
-threonine producing mutant, elevated the specific activity of DDPS 20-fold and
l
-lysine production 2- to 3-fold with concomitant reduction of
l
-threonine in test tube cultures. AL119 containing the
dapA
gene produced 8 g of
l
-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the
dapA
gene shows that it encodes a peptide with an
M
r
of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the
dapA
genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the
dapA
genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by
l
-lysine. The similarity in the amino acid sequences of DDPS in
M. glycogenes
and other organisms suggests that the mutation of the
dapA
gene in DHL122 is located in the region concerned with interaction of the allosteric effector,
l
-lysine.
Role of Fatty Acid De Novo Biosynthesis in Polyhydroxyalkanoic Acid (PHA) and Rhamnolipid Synthesis by Pseudomonads: Establishment of the Transacylase (PhaG)-Mediated Pathway for PHA Biosynthesis in
Escherichia coli
Authors: Rehm Bernd H. A. , Mitsky Timothy A. , Steinbüchel Alexander
Published: July 2002

Since
Pseudomonas aeruginosa
is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various
fab
mutants from
P. aeruginosa
with respect to biosynthesis of PHAs and rhamnolipids. All isogenic
fabA, fabB, fabI, rhlG
, and
phaG
mutants from
P. aeruginosa
showed decreased PHA accumulation and rhamnolipid production. In the
phaG
(encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of
phaG
from
Pseudomonas putida
and expression of the β-ketoacyl reductase gene
rhlG
from
P. aeruginosa
in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn
5
mutants of
P. putida
strongly impaired in PHA production from gluconate. All mutants were complemented by the
phaG
gene from
P. putida
, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant
E. coli
, coexpressing the class II PHA synthase gene
phaC1
together with the
phaG
gene from
P. putida
, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.
Species-Specific PCR for Identification of Common Contaminant Mollicutes in Cell Culture
Authors: Kong Fanrong , James Gregory , Gordon Susanna , Zelynski Anna , Gilbert Gwendolyn L.
Published: July 2002

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale
, and
Acholeplasma laidlawii
are the members of the class
Mollicutes
most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.
Characterization of a
Sinorhizobium
Isolate and Its Extracellular Polymer Implicated in Pollutant Transport in Soil
Authors: Janecka J. , Jenkins M. B. , Brackett N. S. , Lion L. W. , Ghiorse W. C.
Published: July 2002

A bacterium isolated from soil (designated 9702-M4) synthesizes an extracellular polymer that facilitates the transport of such hydrophobic pollutants as polynuclear aromatic hydrocarbons, as well as the toxic metals lead and cadmium in soil. Biolog analysis, growth rate determinations, and percent G+C content identify 9702-M4 as a strain of
Sinorhizobium meliloti
. Sequence analysis of a 16S rDNA fragment gives 9702-M4 a phylogenetic designation most closely related to
Sinorhizobium fredii
. The extracellular polymer of isolate 9702-M4 is composed of both an extracellular polysaccharide (EPS) and a rough lipopolysaccharide. The EPS component is composed mainly of 4-glucose linkages with monomers of galactose, mannose, and glucuronic acid and has pyruval and acetyl constituents. The lipid fraction and the negative charge associated with carbonyl groups of the exopolymer are thought to account for the binding of polynuclear aromatic hydrocarbons and cationic metals.
The Microbial Community Structure of Drinking Water Biofilms Can Be Affected by Phosphorus Availability
Authors: Keinänen Minna M. , Korhonen Leena K. , Lehtola Markku J. , Miettinen Ilkka T. , Martikainen Pertti J. , Vartiainen Terttu , Suutari Merja H.
Published: July 2002

Microbial communities in biofilms grown for 4 and 11 weeks under the flow of drinking water supplemented with 0, 1, 2, and 5 μg of phosphorus liter
−1
and in drinking and warm waters were compared by using phospholipid fatty acids (PLFAs) and lipopolysaccharide 3-hydroxy fatty acids (LPS 3-OH-FAs). Phosphate increased the proportion of PLFAs 16:1ω7
c
and 18:1ω7
c
and affected LPS 3-OH-FAs after 11 weeks of growth, indicating an increase in gram-negative bacteria and changes in their community structure. Differences in community structures between biofilms and drinking and warm waters can be assumed from PLFAs and LPS 3-OH-FAs, concomitantly with adaptive changes in fatty acid chain length, cyclization, and unsaturation.
Expanded Genetic Map of
Gibberella moniliformis
(
Fusarium verticillioides
)
Authors: Jurgenson James E. , Zeller Kurt A. , Leslie John F.
Published: July 2002

Gibberella moniliformis
(
Fusarium verticillioides
) is primarily a pathogen of maize, but it can also cause disease in other crop species. This pathogenicity, as well as the contamination of food- and feedstuffs with the fumonisin mycotoxins, results in economically significant losses to both farmers and food processors. The dissection of important biological characters in this fungus has been hampered by the lack of a uniformly dense genetic map. The existing restriction fragment length polymorphism-based map contains significant gaps, making it difficult to routinely locate biologically important genes, such as those involved in pathogenicity or mycotoxin production, with precision. We utilized amplified fragment length polymorphisms (AFLPs) to saturate the existing genetic map and added 486 AFLP markers to the ∼150 markers on the existing map. The resulting map has an average marker interval of 3.9 map units and averages ∼21 kb/map unit. The additional markers expanded the map from 1,452 to 2,188 map units distributed across 12 chromosomes. The maximum distance between adjacent markers is 29 map units. We identified AFLP markers less than 1 map unit from the mating type (
MAT
) locus and 2.5 map units from the spore killer (
SK
) locus; eight AFLP markers map within 8.5 units of the
FUM1
(fumonisin biosynthetic) locus. The increased saturation of this map will facilitate further development of
G. moniliformis
as a model system for the genetic and population genetic studies of related, but less genetically tractable, plant pathogenic fungi.
Serotype-Cross-Reactive Immunoglobulin M Responses in Dengue Virus Infections Determined by Enzyme-Linked Immunosorbent Assay
Authors: Nawa Masaru , Yamada Ken-Ichiro , Takasaki Tomohiko , Akatsuka Toshitaka , Kurane Ichiro
Published: July 2002

We developed immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assays (ELISAs) with four monovalent dengue virus antigens. We attempted to determine whether IgM responses in dengue virus infections are serotype specific or serotype cross-reactive. Serum samples from 14 confirmed dengue cases were examined. In these 14 cases, which consisted of 12 Japanese and 2 non-Japanese patients, infecting dengue virus serotypes were defined by reverse transcription-PCR. Thirteen of the 14 cases were IgM positive in ELISA. IgM responses were serotype cross-reactive in these 13 cases but were highest against infecting dengue virus serotype in 9 of the 13 cases. These results indicate that IgM responses are generally dengue serotype cross-reactive but that IgM levels are highest against the infecting serotype in most dengue cases.
Procalcitonin Does Not Discriminate Infection from Inflammation after Allogeneic Bone Marrow Transplantation
Authors: Blijlevens N. M. A. , Donnelly J. P. , Meis J. F. G. M. , De Keizer M. H. , De Pauw B. E.
Published: July 2002

Procalcitonin (PCT) is an early marker of bacterial infection but little is known about its value in neutropenic allogeneic bone marrow transplant (BMT) recipients. We collected plasma from 12 recipients of T-cell-depleted HLA-matched related BMT recipients who had been treated preemptively with meropenem from the day after BMT for at least 15 days. PCT and C-reactive protein (CRP) concentrations were determined on BMT days 1, 5, 8, 12, and 15, and their relationship to inflammatory events (IE), including mucositis, microbiologically and clinically defined infections, acute graft-versus-host disease (GVDH), and unexplained fever, was then determined. The PCT concentrations were all low and never exceeded 4 μg/liter, unlike CRP concentrations, which spanned the full range up to 350 mg/liter. All patients had mucositis, and there was no significant difference between PCT concentrations associated with mucositis alone and those associated with an additional IE on BMT days 1 to 12. However, on BMT day 15, the mean concentrations of PCT were 0.37 ± 0.05 μg/liter for the 10 patients that had an additional IE, compared with 0.11 ± 0.03 μg/liter for the 2 patients with mucositis only (
P
= 0.012), and GVHD rather than infection was involved in six cases. PCT was also not a sensitive marker of gram-positive bacteremia or pulmonary aspergillosis. Thus, PCT is of little value in discriminating infections from other inflammatory complications that occur following allogeneic BMT.
Assessment of Neutrophil Function in Patients with Septic Shock: Comparison of Methods
Authors: Wenisch C. , Fladerer P. , Patruta S. , Krause R. , Hörl W.
Published: July 2002

Patients with septic shock are shown to have decreased neutrophil phagocytic function by multiple assays, and their assessment by whole-blood assays (fluorescence-activated cell sorter analysis) correlates with assays requiring isolated neutrophils (microscopic and spectrophotometric assays). For patients with similar underlying conditions but without septic shock, this correlation does not occur.
Sacbrood Virus of the Honeybee (
Apis mellifera
): Rapid Identification and Phylogenetic Analysis Using Reverse Transcription-PCR
Authors: Grabensteiner Elvira , Ritter Wolfgang , Carter Michael J. , Davison Sean , Pechhacker Hermann , Kolodziejek Jolanta , Boecking Otto , Derakhshifar Irmgard , Moosbeckhofer Rudolf , Licek Elisabeth , Nowotny Norbert
Published: July 2002

Sacbrood virus (SBV) infects larvae of the honeybee (
Apis mellifera
), resulting in failure to pupate and death. Until now, identification of viruses in honeybee infections has been based on traditional methods such as electron microscopy, immunodiffusion, and enzyme-linked immunosorbent assay. Culture cannot be used because no honeybee cell lines are available. These techniques are low in sensitivity and specificity. However, the complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a reverse transcription-PCR (RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. RT-PCR was used to target five different areas of the SBV genome using infected honeybees and larvae originating from geographically distinct regions. The RT-PCR assay proved to be a rapid, specific, and sensitive diagnostic tool for the direct detection of SBV nucleic acid in samples of infected honeybees and brood regardless of geographic origin. The amplification products were sequenced, and phylogenetic analysis suggested the existence of at least three distinct genotypes of SBV.
Real-Time PCR as a New Tool for Quantifying
Leishmania infantum
in Liver in Infected Mice
Authors: Bretagne Stéphane , Durand Rémy , Olivi Martine , Garin Jean-François , Sulahian Annie , Rivollet Danièle , Vidaud Michel , Deniau Michèle
Published: July 2002

The parasitic loads of mouse livers experimentally infected with
Leishmania infantum
were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. The
Leishmania
DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.
High Serum Interleukin-10 and Tumor Necrosis Factor Alpha Levels in Chronic Paracoccidioidomycosis
Authors: Fornari M. C. , Bava A. J. , Guereño M. T. , Berardi V. E. , Silaf M. R. , Negroni R. , Diez R. A.
Published: July 2002

In patients with chronic paracoccidioidomycosis (
n
= 10), levels of tumor necrosis factor alpha, interleukin-10, and interleukin-2 in serum, measured by enzyme-linked immunosorbent assay (in picograms per milliliter, as mean ± standard error of the mean), were higher than in normal controls (
n
= 8): 186 ± 40 versus 40 ± 7 (
P
< 0.05), 203 ± 95 versus 20 ± 8 (
P
= 0.001), and 96.3 ± 78.57 versus 1.19 ± 1.19 (
P
= 0.045), respectively. Gamma interferon and interleukin-4 levels were similar in patients and controls.
Presence of Oligoclonal T Cells in Cerebrospinal Fluid of a Child with Multiphasic Disseminated Encephalomyelitis following Hepatitis A Virus Infection
Authors: Oleszak Emilia L. , Lin Wan Lu , Legido Agustin , Melvin Joseph , Hardison Huntley , Hoffman Brad E. , Katsetos Christos D. , Platsoucas Chris D.
Published: July 2002

We have investigated the clonality of β-chain T-cell receptor (TCR) transcripts from the cerebrospinal fluid (CSF) and peripheral blood from a 7-year old child who developed a multiphasic disseminated encephalomyelitis following an infection with hepatitis A virus. We amplified β-chain TCR transcripts by nonpalindromic adaptor (NPA)-PCR–Vβ-specific PCR. TCR transcripts from only five Vβ families (Vβ13, Vβ3, Vβ17, Vβ8, and Vβ20) were detected in CSF. The amplified products were combined, cloned, and sequenced. Sequence analysis revealed in the CSF substantial proportions of identical β-chain of TCR transcripts, demonstrating oligoclonal populations of T cells. Seventeen of 35 (48%) transcripts were 100% identical, demonstrating a major Vβ13.3 Dβ2.1 Jβ1.3 clonal expansion. Six of 35 (17%) transcripts were also 100% identical, revealing a second Vβ13 clonal expansion (Vβ13.1 Dβ2.1 Jβ1.2). Clonal expansions were also found within the Vβ3 family (transcript Vβ3.1 Dβ2.1 Jβ1.5 accounted for 5 of 35 transcripts [14%]) and within the Vβ20 family (transcript Vβ20.1 Dβ1.1 Jβ2.4 accounted for 3 of 35 transcripts [8%]). These results demonstrate the presence of T-cell oligoclonal expansions in the CSF of this patient following infection with hepatitis A virus. Analysis of the CDR3 motifs revealed that two of the clonally expanded T-cell clones exhibited substantial homology to myelin basic protein-reactive T-cell clones. In contrast, all Vβ TCR families were expressed in peripheral blood lymphocytes. Oligoclonal expansions of T cells were not detected in the peripheral blood of this patient. It remains to be determined whether these clonally expanded T cells are specific for hepatitis A viral antigen(s) or host central nervous system antigen(s) and whether molecular mimicry between hepatitis A viral protein and a host protein is responsible for demyelinating disease in this patient.
Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants
Authors: Neri Simona , Mariani Erminia , Meneghetti Alessandra , Cattini Luca , Facchini Andrea
Published: July 2002

Cytotoxicity assays provide an in vitro evaluation of the lytic activity of NK and T cells against tumors or transformed cells. However, none of these methods allow the recovery of cells or supernatants after the assay. We standardized a microcytotoxicity test using calcein-acetoxymethyl (calcein-AM) dye that requires very small quantities of cells while maintaining the same sensitivity as the traditional51Cr assay. The assay is applicable to resting as well as activated human effector cells and uses different targets such as human cell lines that are adherent or growing in suspension and resistant or sensitive. The most important feature of the method is the possibility of recovering cells and supernatants for additional analyses such as phenotyping and evaluation of soluble factors.
Lactoferrin Protects against Development of Hepatitis Caused by Sensitization of Kupffer Cells by Lipopolysaccharide
Authors: Yamaguchi Makoto , Matsuura Motoi , Kobayashi Kiyoshi , Sasaki Hajime , Yajima Takaji , Kuwata Tamotsu
Published: July 2002

BALB/c mice were intravenously injected with lipopolysaccharide (LPS) (0.05 μg/g of body weight) 7 days after being primed with zymosan. Recombinant human lactoferrin (250 μg/g of body weight), intravenously administered 1 day before the injection of LPS, significantly lessened the severity of hepatitis, as assessed by levels of serum alanine transaminase compared to those seen when casein was administered. The transient rise of serum tumor necrosis factor alpha (TNF-α) after LPS treatment was also significantly lowered by the intravenous administration of lactoferrin, suggesting that the effect of lactoferrin was due to the suppression of TNF-α production. The following results indicate that the sites of action of lactoferrin for the suppression of the development of this type of hepatitis are Kupffer cells. Gadolinium chloride, a substance known to eliminate Kupffer cells, administered 1 day before LPS, inhibited the transient rise of TNF-α and protected against the development of hepatitis. Kupffer cells isolated from mice intraperitoneally injected with recombinant human lactoferrin became refractory to LPS. The specific interaction of recombinant human lactoferrin with the Kupffer cells was shown by a binding assay, which revealed two types of binding sites on mouse Kupffer cells. Of the two dissociation constants determined in this way, the lower dissociation constant, 0.47 × 10−6M, was within the range of the 50% effective doses for the suppression of TNF-α production. These results suggest that recombinant human lactoferrin administered to mice suppresses the production of TNF-α by Kupffer cells by directly associating with the binding sites on these cells.
Grape Seed Extract Activates Th1 Cells In Vitro
Authors: Nair Narayanan , Mahajan Supriya , Chawda Ram , Kandaswami Chithan , Shanahan Thomas C. , Schwartz Stanley A.
Published: July 2002

Although flavonoids manifest a diverse range of biological activities, including antitumor and antiviral effects, the molecular mechanisms underlying these activities await elucidation. We hypothesize that the flavonoid constituents of a proprietary grape seed extract (GSE) that contains procyandins exert significant antiviral and antitumor effects, by inducing production of the Th1-derived cytokine gamma interferon (IFN-γ) by peripheral blood mononuclear cells) from healthy donors. Our results show that GSE significantly induced the transcription of IFN-γ mRNA as demonstrated by reverse transcription-PCR but had no effect on the Th2-derived cytokine interleukin-6. The enhancing effect of GSE on IFN-γ expression was further supported by a concomitant increase in the number of cells with intracytoplasmic IFN-γ as well as the synthesis and secretion of IFN-γ. Our results demonstrate that the potentially beneficial immunostimulatory effects of GSE may be mediated through the induction of IFN-γ.
Epidemiology and Clinical Features ofCryptosporidiumInfection in Immunocompromised Patients
Authors: Hunter Paul R. , Nichols Gordon
Published: July 2002

Cryptosporidium spp. are a major cause of diarrheal disease in both immunocompetent and immunodeficient individuals. They also cause waterborne disease in both the United States and United Kingdom. Studies on the mechanisms of immunity to cryptosporidiosis indicate the importance of the T-cell response. The spectrum and severity of disease in immunocompromised individuals with cryptosporidiosis reflect this importance since the most severe disease is seen in individuals with defects in the T-cell response. The most commonly studied group is that of patients with AIDS. These patients suffer from more severe and prolonged gastrointestinal disease that can be fatal; in addition, body systems other than the gastrointestinal tract may be affected. The widespread use of antiretroviral therapy does appear to be having a beneficial effect on recovery from cryptosporidiosis and on the frequency of infection in human immunodeficiency virus-positive patients. Other diseases that are associated with increased risk of severe cryptosporidiosis, such as primary immunodeficiencies, most notably severe combined immunodeficiency syndrome, are also predominantly associated with T-cell defects. Of the remaining groups, children with acute leukemia seem to be most at risk from cryptosporidiosis. There is less evidence of severe complications in patients with other malignant diseases or in those receiving immunosuppressive chemotherapy.
Integrated Approach to Malaria Control
Authors: Shiff Clive
Published: July 2002

Malaria draws global attention in a cyclic manner, with interest and associated financing waxing and waning according to political and humanitarian concerns. Currently we are on an upswing, which should be carefully developed. Malaria parasites have been eliminated from Europe and North America through the use of residual insecticides and manipulation of environmental and ecological characteristics; however, in many tropical and some temperate areas the incidence of disease is increasing dramatically. Much of this increase results from a breakdown of effective control methods developed and implemented in the 1960s, but it has also occurred because of a lack of trained scientists and control specialists who live and work in the areas of endemic infection. Add to this the widespread resistance to the most effective antimalarial drug, chloroquine, developing resistance to other first-line drugs such as sulfadoxine-pyrimethamine, and resistance of certain vector species of mosquito to some of the previously effective insecticides and we have a crisis situation. Vaccine research has proceeded for over 30 years, but as yet there is no effective product, although research continues in many promising areas. A global strategy for malaria control has been accepted, but there are critics who suggest that the single strategy cannot confront the wide range of conditions in which malaria exists and that reliance on chemotherapy without proper control of drug usage and diagnosis will select for drug resistant parasites, thus exacerbating the problem. An integrated approach to control using vector control strategies based on the biology of the mosquito, the epidemiology of the parasite, and human behavior patterns is needed to prevent continued upsurge in malaria in the endemic areas.
Identification of 11 pH-Regulated Genes in
Borrelia burgdorferi
Localizing to Linear Plasmids
Authors: Carroll James A. , Cordova Rebecca M. , Garon Claude F.
Published: July 2002

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.
Immunopathologic Effects of Tumor Necrosis Factor Alpha in Murine Mycobacterial Infection Are Dose Dependent
Authors: Bekker Linda-Gail , Moreira Andre L. , Bergtold Amy , Freeman Sherry , Ryffel Bernard , Kaplan Gilla
Published: July 2002

In experimental mycobacterial infection, tumor necrosis factor alpha (TNF-α) is required for control of bacillary growth and the protective granulomatous response, but may cause immunopathology. To directly examine the positive and detrimental effects of this cytokine, a murine model was used in which different amounts of TNF-α were delivered to the site of infection. Mice with a disruption in the TNF-α gene (TNF-KO) or wild-type mice were infected with low or high doses of recombinantMycobacterium bovisBCG that secreted murine TNF-α (BCG-TNF). Infection of TNF-KO mice with BCG containing the vector (BCG-vector) at a low dose led to increased bacillary load in all organs and an extensive granulomatous response in the lungs and spleen. The mice succumbed to the infection by ∼40 days. However, when TNF-KO mice were infected with low doses of BCG-TNF, bacillary growth was controlled, granulomas were small and well differentiated, the spleen was not enlarged, and the mice survived. Infection with high inocula of BCG-TNF resulted in bacterial clearance, but was accompanied by severe inflammation in the lungs and spleen and earlier death compared to the results from the mice infected with high inocula of BCG-vector. Wild-type mice controlled infection with either recombinant strain, but showed decreased survival following high-dose BCG-TNF infection. The effects of TNF-α required signaling through an intact receptor, since the differential effects were not observed when TNF-α receptor-deficient mice were infected. The results suggest that the relative amount of TNF-α at the site of infection determines whether the cytokine is protective or destructive.