bluuebunny/crossref_metadata_2025 huggingface dataset:
Pineal expression-promoting element (PIPE), a
cis-
acting element, directs pineal-specific gene expression in zebrafish
Authors: Asaoka Yoichi , Mano Hiroaki , Kojima Daisuke , Fukada Yoshitaka
Published: November 2002

The pineal gland, sharing morphological and biochemical similarities with the retina, plays a unique and central role in the photoneuroendocrine system. The unique development of the pineal gland is directed by a specific combination of the expressed genes, but little is known about the regulatory mechanism underlying the pineal-specific gene expression. We isolated a 1.1-kbp fragment upstream of the zebrafish exo-rhodopsin (
exorh
) gene, which is expressed specifically in the pineal gland. Transgenic analysis using an enhanced green fluorescent protein reporter gene demonstrated that the proximal 147-bp region of the
exorh
promoter is sufficient to direct pineal-specific expression. This region contains three copies of a putative cone rod homeobox (Crx)/Otx-binding site, which is known to be required for expression of both retina- and pineal-specific genes. Deletion and mutational analyses of the
exorh
promoter revealed that a previously uncharacterized sequence TGACCCCAATCT termed pineal expression-promoting element (PIPE) is required for pineal-specific promoter activity in addition to the Crx/Otx-binding sites. By using the zebrafish rhodopsin (
rh
) promoter that drives retina-specific expression, we created a reporter construct having ectopic PIPE in the
rh
promoter at a position equivalent to that in the
exorh
promoter by introducing five nucleotide changes. Such a slight modification in the
rh
promoter induced ectopic enhanced green fluorescent protein expression in the pineal gland without affecting its retinal expression. These results identify PIPE as a critical
cis
-element contributing to the pineal-specific gene expression, in combination with the Crx/Otx-binding site(s).
Measurement
in vivo
of proliferation rates of slow turnover cells by
2
H
2
O labeling of the deoxyribose moiety of DNA
Authors: Neese R. A. , Misell L. M. , Turner S. , Chu A. , Kim J. , Cesar D. , Hoh R. , Antelo F. , Strawford A. , McCune J. M. , Christiansen M. , Hellerstein M. K.
Published: November 2002

We describe here a method for measuring DNA replication and, thus, cell proliferation in slow turnover cells that is suitable for use in humans. The technique is based on the incorporation of
2
H
2
O into the deoxyribose (dR) moiety of purine deoxyribonucleotides in dividing cells. For initial validation, rodents were administered 4%
2
H
2
O in drinking water. The proliferation rate of mammary epithelial cells in mice was 2.9% per day and increased 5-fold during pregnancy. Administration of estradiol pellets (0–200 μg) to ovariectomized rats increased mammary epithelial cell proliferation, according to a dose–response relationship up to the 100 μg dose. Similarly, proliferation of colon epithelial cells was stimulated in a dose–response manner by dietary cholic acid in rats. Bromodeoxyuridine labeling correlated with the
2
H
2
O results. Proliferation of slow turnover cells was then measured. Vascular smooth muscle cells isolated from mouse aorta divided with a half-life in the range of 270–400 days and die-away values after
2
H
2
O wash-out confirmed these slow turnover rates. The proliferation rate of an adipocyte-enriched fraction from mouse adipose tissue depots was 1–1.5% new cells per day, whereas obese ad libitum-fed ob/ob mice exhibited markedly higher fractional and absolute proliferation rates. In humans, stable long-term
2
H
2
O enrichments in body water were achieved by daily
2
H
2
O intake, without toxicities. Labeled dR from fully turned-over blood cells (monocytes or granulocytes) exhibited a consistent amplification factor relative to body
2
H
2
O enrichment (≈3.5-fold). The fraction of newly divided naive-phenotype T cells after 9 weeks of labeling with
2
H
2
O was 0.056 (CD4
+
) and 0.043 (CD8
+
) (replacement rate <0.1% per day). In summary,
2
H
2
O labeling of dR in DNA allows safe, convenient, reproducible, and inexpensive measurement of cell proliferation in humans and experimental animals and is well suited for slow turnover cells.
Killing of
Caenorhabditis
elegans
by
Cryptococcus neoformans
as a model of yeast pathogenesis
Authors: Mylonakis Eleftherios , Ausubel Frederick M. , Perfect John R. , Heitman Joseph , Calderwood Stephen B.
Published: November 2002

We found that the well-studied nematode
Caenorhabditis elegans
can use various yeasts, including
Cryptococcus laurentii
and
Cryptococcus kuetzingii
, as a sole source of food, producing similar brood sizes compared with growth on its usual laboratory food source
Escherichia coli
OP50.
C. elegans
grown on these yeasts had a life span similar to (
C. laurentii
) or longer than (
C. kuetzingii
) those fed on
E. coli
. However, the human pathogenic yeast
Cryptococcus neoformans
killed
C. elegans
, and the
C. neoformans
polysaccharide capsule as well as several
C. neoformans
genes previously shown to be involved in mammalian virulence were also shown to play a role in
C. elegans
killing. These included genes associated with signal transduction pathways (
GPA1
,
PKA1
,
PKR1
, and
RAS1
), laccase production (
LAC1
), and the α mating type.
C. neoformans
adenine auxotrophs, which are less virulent in mammals, were also less virulent in
C. elegans
. These results support the model that mammalian pathogenesis of
C. neoformans
may be a consequence of adaptations that have evolved during the interaction of
C. neoformans
with environmental predators such as free-living nematodes and amoebae and suggest that
C. elegans
can be used as a simple model host in which
C. neoformans
pathogenesis can be readily studied.
The systemin precursor gene regulates both defensive and developmental genes in
Solanum tuberosum
Authors: Narváez-Vásquez Javier , Ryan Clarence A.
Published: November 2002

Transformation of
Solanum tuberosum
, cv. Desiree, with the tomato prosystemin gene, regulated by the 35S cauliflower mosaic virus promoter, resulted in constitutive increase in defensive proteins in potato leaves, similar to its effects in tomato plants, but also resulted in a dramatic increase in storage protein levels in potato tubers. Tubers from selected transformed lines contained 4- to 5-fold increases in proteinase inhibitor I and II proteins, >50% more soluble and dry weight protein, and >50% more total nitrogen and total free amino acids than found in wild-type tubers. These results suggest that the prosystemin gene plays a dual role in potato plants in regulating proteinase inhibitor synthesis in leaves in response to wounding and in regulating storage protein synthesis in potato tubers in response to developmental cues. The results indicated that components of the systemin signaling pathway normally found in leaves have been recruited by potato plants to be developmentally regulated to synthesize and accumulate large quantities of storage proteins in tubers.
Neurovestibular modulation of circadian and homeostatic regulation: Vestibulohypothalamic connection?
Authors: Fuller Patrick M. , Jones Timothy A. , Jones Sherri M. , Fuller Charles A.
Published: November 2002

Chronic exposure to increased force environments (+G) has pronounced effects on the circadian and homeostatic regulation of body temperature (T
b
), ambulatory activity (Act), heart rate, feeding, and adiposity. By using the Brn 3.1 knockout mouse, which lacks vestibular hair cells, we recently described a major role of the vestibular system in mediating some of these adaptive responses. The present study used the C57BL/6JE
i-het
mouse strain (
het
), which lacks macular otoconia, to elucidate the contribution of specific vestibular receptors. In this study, eight
het
and eight WT mice were exposed to 2G for 8 weeks by means of chronic centrifugation. In addition, eight
het
and eight WT mice were maintained as 1G controls in similar conditions. Upon 2G exposure, the WT exhibited a decrease in T
b
and an attenuated T
b
circadian rhythm. Act means and rhythms also were attenuated. Body mass and food intake were significantly lower than the 1G controls. After 8 weeks, percent body fat was significantly lower in the WT mice (
P
< 0.0001). In contrast, the
het
mice did not exhibit a decrease in mean T
b
and only a slight decrease in T
b
circadian amplitude.
het
Act levels were attenuated similarly to the WT mice. Body mass and food intake were only slightly attenuated in the
het
mice, and percent body fat, after 8 weeks, was not different in the 2G
het
group. These results link the vestibular macular receptors with specific alterations in homeostatic and circadian regulation.
Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland
Authors: Postiglione M. P. , Parlato R. , Rodriguez-Mallon A. , Rosica A. , Mithbaokar P. , Maresca M. , Marians R. C. , Davies T. F. , Zannini M. S. , De Felice M. , Di Lauro R.
Published: November 2002

The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSH/TSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (

pit
dw

/

pit
dw

) or of a functional TSHR (

tshr
hyt

/

tshr
hyt

and
TSHR
-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid.
A simple alfalfa seedling infection model for
Pseudomonas aeruginosa
strains associated with cystic fibrosis shows AlgT (sigma-22) and RhlR contribute to pathogenesis
Authors: Silo-Suh Laura , Suh Sang-Jin , Sokol Pamela A. , Ohman Dennis E.
Published: November 2002

A sensitive plant infection model was developed to identify virulence factors in nontypeable, alginate overproducing (mucoid)
Pseudomonas aeruginosa
strains isolated from cystic fibrosis (CF) patients with chronic pulmonary disease. Nontypeable strains with defects in lipopolysaccharide O-side chains are common to CF and often exhibit low virulence in animal models of infection. However, 1,000 such bacteria were enough to show disease symptoms in the alfalfa infection. A typical mucoid CF isolate, FRD1, and its isogenic mutants were tested for alfalfa seedling infection. Although defects in the global regulators Vfr, RpoS, PvdS, or LasR had no discernable effect on virulence, a defect in RhlR reduced the infection frequency by >50%. A defect in alginate biosynthesis resulted in plant disease with >3-fold more bacteria per plant, suggesting that alginate overproduction attenuated bacterial growth
in planta
. FRD1 derivatives lacking AlgT, a sigma factor required for alginate production, were reduced >50% in the frequency of infection. Thus, AlgT apparently regulates factors in FRD1, besides alginate, important for pathogenesis. In contrast, in a non-CF strain, PAO1, an
algT
mutation did not affect its virulence on alfalfa. Conversely, PAO1 virulence was reduced in a
mucA
mutant that overproduced alginate. These observations suggested that mucoid conversion in CF may be driven by a selection for organisms with attenuated virulence or growth in the lung, which promotes a chronic infection. These studies also demonstrated that the wounded alfalfa seedling infection model is a useful tool to identify factors contributing to the persistence of
P. aeruginosa
in CF.
Fast synaptic transmission between striatal spiny projection neurons
Authors: Czubayko Uwe , Plenz Dietmar
Published: November 2002

Striatal inhibition plays an important role in models of cortex-basal ganglia function and is altered in many basal ganglia diseases. The γ-aminobutyric acid ergic spiny projection neuron comprises >95% of striatal neurons, but despite strong anatomical evidence, the electrophysiological properties and functions of their local axon collaterals are unknown. We simultaneously recorded from adjacent spiny projection neurons (<5–10 μm) in whole-cell patch mode and demonstrated a fast synaptic connection between 26/69 pairs in cortex-striatum-substantia nigra organotypic cultures and 5/38 pairs in acute striatal slices. The synapse, which was blocked by γ-aminobutyric acid type A antagonists, displayed a wide range of failure rates, was depolarizing at rest, and reversed above −60 mV. Presynaptic bursts of action potentials were highly correlated with total postsynaptic depolarization at rest. Synaptic transmission was optimized for burst discharge >14 Hz and showed considerable short-term plasticity, including paired-pulse depression at intervals <25 ms, intraburst facilitation, and interburst augmentation. This activity-dependent collateral interaction provides the basis for a new class of basal ganglia models in which striatal neurons cooperate as well as compete during processing of cortical inputs.
An allelic series of mutations in
Smad2
and
Smad4
identified in a genotype-based screen of
N
-ethyl-
N
- nitrosourea-mutagenized mouse embryonic stem cells
Authors: Vivian Jay L. , Chen Yijing , Yee Della , Schneider Elizabeth , Magnuson Terry
Published: November 2002

Using selectable genes as proof of principle, a new high-throughput genotype-based mutation screen in mouse embryonic stem (ES) cells was developed [Chen
et al.
(2002)
Nat. Genet.
24, 314–317]. If expanded to nonselectable genes, this approach would allow one to proceed quickly from sequence to whole-animal phenotypes. Here data are presented showing that a screen of a cryopreserved library of clonal, germ line competent,
N
-ethyl-
N
-nitrosurea (ENU) mutagenized ES cells can identify a large series of allelic mutations in
Smad2
and
Smad4
, two nonselectable genes of the transforming growth factor β superfamily of signaling molecules. Whole animal phenotypic analyses of some of these alleles provided evidence for novel developmental processes mediated by these components of transforming growth factor β signaling, demonstrating the utility of non-null alleles created by chemical mutagens. The accurately assessed mutation load of the ES cell library indicates that it is a valuable resource for developing mouse lines for genetic and functional studies. This methodology can conceptually be applied for the generation of an allelic series of subtle mutations at any locus of interest in the mouse.
Bombyxin is a growth factor for wing imaginal disks in Lepidoptera
Authors: Nijhout H. Frederik , Grunert Laura W.
Published: November 2002

The mechanisms that control the growth rate of internal tissues during postembryonic development are poorly understood. In insects, the growth rate of imaginal disks varies with nutrition and keeps pace with variation in somatic growth. We describe here a mechanism by which the growth of wing imaginal disks is controlled. When wing imaginal disks of the butterflyPrecis coeniaare removed from the larva and placed in a standard nutrient-rich tissue culture medium they stop growing, suggesting that nutrients alone are not sufficient to support normal growth. Such disks can be made to grow at a normal rate by supplementing the culture medium with an optimal concentration of the steroid hormone 20-hydroxyecdysone and with hemolymph taken from growing larvae. The growth-promoting activity of the hemolymph is caused by a heat-stable factor that can be extracted from the CNS and appears to be identical to the neurohormone bombyxin, a member of the insulin family of proteins. Synthetic bombyxin stimulates growth at concentrations as low as 30 ng/ml, and specific antibodies to bombyxin completely remove growth-promoting activity from the hemolymph. Bombyxin evidently acts together with 20-hydroxyecdysone to stimulate cell division and growth of wing imaginal disks. It appears that the level of bombyxin in the hemolymph is modulated by the brain in response to variation in nutrition and is part of the mechanism that coordinates the growth of internal organs with overall somatic growth.
Measurements of endothelial cell-to-cell and cell-to-substrate gaps and micromechanical properties of endothelial cells during monocyte adhesion
Authors: Kataoka Noriyuki , Iwaki Kanso , Hashimoto Ken , Mochizuki Seiichi , Ogasawara Yasuo , Sato Masaaki , Tsujioka Katsuhiko , Kajiya Fumihiko
Published: November 2002

The interaction between monocytes and endothelial cells is considered to play a major role in the early stage of atherosclerosis, and the involved endothelial cell micromechanics may provide us with important aspects of atherogenesis. In the present study, we evaluated (i) the endothelial cell-to-cell and cell-to-substrate gaps with the electric cell-substrate impedance sensing system, which can detect the nanometer order changes of cell-to-cell and cell-to-substrate distances separately, and (ii) the endothelial cell micromechanical properties with an atomic force microscope after application of monocytes to endothelial cells. Application of monocytic THP-1 cells to IL-1β-stimulated human umbilical vein endothelial cells immediately decreased the electrical resistance of the endothelial cell-to-substrate (increase of the cell-to-substrate gap), whereas the endothelial cell-to-cell resistance (cell-to-cell gap) did not change. The elastic modulus of the endothelial cells decreased after 2-h monocyte application, indicating an increase of endothelial cell deformability. In conclusion, the interaction of the monocytes to the endothelial cells reduced the adhesiveness to the substrate and increased the deformability of endothelial cells. These changes in the adhesiveness and the deformability may facilitate migration of monocytes, a key process of atherogenesis in the later stage.
The signal of ancient introns is obscured by intron density and homolog number
Authors: Roy Scott William , Fedorov Alexei , Gilbert Walter
Published: November 2002

In ancient genes whose products have known 3-dimensional structures, an excess of phase zero introns (those that lie between the codons) appear in the boundaries of modules, compact regions of the polypeptide chain. These excesses are highly significant and could support the hypothesis that ancient genes were assembled by exon shuffling involving compact modules. (Phase one and two introns, and many phase zero introns, appear to arise later.) However, as more genes, with larger numbers of homologs and intron positions, were examined, the effects became smaller, dropping from a 40% excess to an 8% excess as the number of intron positions increased from 570 to 3,328, even though the statistical significance remained strong. An interpretation of this behavior is that novel inserted positions appearing in homologs washed out the signal from a finite number of ancient positions. Here we show that this is likely to be the case. Analyses of intron positions restricted to those in genes for which relatively few intron positions from homologs are known, or to those in genes with a small number of known homologous gene structures, show a significant correlation of phase zero intron positions with the module structure, which weakens as the density of attributed intron positions or the number of homologs increases. These effects do not appear for phase one and phase two introns. This finding matches the expectation of the mixed model of intron origin, in which a fraction of phase zero introns are left from the assembly of the first genes, while other introns have been added in the course of evolution.
Ultrafast surface hydration dynamics and expression of protein functionality: α-Chymotrypsin
Authors: Pal Samir Kumar , Peon Jorge , Zewail Ahmed H.
Published: November 2002

We report studies of hydration dynamics at the surface of the enzyme protein bovine pancreatic α-chymotrypsin. The probe is the well known 1-anilinonaphthalene-8-sulfonate, which binds selectively in the native state of the protein, not the molten globule, as shown by x-ray crystallography. With femtosecond time resolution, we examined the hydration dynamics at two pHs, when the protein is physiologically in the inactive state (pH 3.6) or the active state (pH 6.7); the global structure and the binding site remain the same. The hydration correlation function,
C
(
t
), whose decay is governed by the rotational and translational motions of water molecules at the site, shows the behavior observed in this laboratory for other proteins, Subtilisin
Carlsberg
and Monellin, using the intrinsic amino acid tryptophan as a probe for surface hydration. However, the time scales and amplitudes vary drastically at the two pHs. For the inactive protein state,
C
(
t
) decays with an ultrafast component, close to bulk-type behavior, but 50% of the
C
(
t
) decays at a much slower rate, τ = 43 ps. In contrast, for the active state, the ultrafast component becomes dominant (90%) and the slow component changes to a faster decay, τ = 28 ps. These results indicate that in the active state water molecules in the hydration layer around the site have a high degree of mobility, whereas in the inactive state the water is more rigidly structured. For the substrate–enzyme complex, the function and dynamics at the probe site are correlated, and the relevance to the enzymatic action is clear.
An exchanger-like protein underlies the large Mg
2+
current in
Paramecium
Authors: Haynes W. John , Kung Ching , Saimi Yoshiro , Preston Robin R.
Published: November 2002

There are very few molecules known to transport Mg
2+
in eukaryotes. The membrane of
Paramecium tetraurelia
passes a large Mg
2+
-selective current and exhibits a corresponding backward swimming behavior. Both are missing in a group of mutants called
eccentric
. By sorting an indexed WT genomic library through microinjection into the macronucleus, we have isolated a DNA fragment that complements the
eccentric
mutations. The Mg
2+
currents and behavior are restored fully in the transformed cells. Surprisingly, the conceptually translated protein is not homologous to any known ion channel but instead has some similarity to K
+
-dependent Na
+
/Ca
2+
exchangers. Exchangers are either electrically silent or only pass very small and slow currents compared with ion-channel currents. In light of recent ion-channel crystal structures and considering the need to have narrow ion-selective filters, we speculate on how an exchanger might evolve to show channel-like activities in special circumstances. The significance of finding the molecular basis of a Mg
2+
-specific pathway is also discussed.
Reorganization of the cochleotopic map in the bat's auditory system by inhibition
Authors: Xiao Zhongju , Suga Nobuo
Published: November 2002

The central auditory system of the mustached bat shows two types of reorganization of cochleotopic (frequency) maps: expanded reorganization resulting from shifts in the best frequencies (BFs) of neurons toward the BF of repetitively stimulated cortical neurons (hereafter centripetal BF shifts) and compressed reorganization resulting from the BF shifts of neurons away from the BF of the stimulated cortical neurons (hereafter centrifugal BF shifts). Facilitation and inhibition evoked by the corticofugal system have been hypothesized to be respectively related to centripetal and centrifugal BF shifts. If this hypothesis is correct, bicuculline (an antagonist of inhibitory GABA-A receptors) applied to cortical neurons would change centrifugal BF shifts into centripetal BF shifts. In the mustached bat, electric stimulation of cortical Doppler-shifted constant-frequency neurons, which are highly specialized for frequency analysis, evokes the centrifugal BF shifts of ipsilateral collicular and cortical Doppler-shifted constant-frequency neurons and contralateral cochlear hair cells. Bicuculline applied to the stimulation site changed the centrifugal BF shifts into centripetal BF shifts. On the other hand, electric stimulation of neurons in the posterior division of the auditory cortex, which are not particularly specialized for frequency analysis, evokes centripetal BF shifts of cortical neurons located near the stimulated cortical neurons. Bicuculline applied to the stimulation site augmented centripetal BF shifts but did not change the direction of the shifts. These observations support the hypothesis and indicate that centripetal and centrifugal BF shifts are both based on a single mechanism consisting of two components: facilitation and inhibition.
A comparison of the molecular clock of hepatitis C virus in the United States and Japan predicts that hepatocellular carcinoma incidence in the United States will increase over the next two decades
Authors: Tanaka Yasuhito , Hanada Kousuke , Mizokami Masashi , Yeo Anthony E. T. , Shih J. Wai-Kuo , Gojobori Takashi , Alter Harvey J.
Published: November 2002

The prevalence of hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) is considerably lower in the U.S. than in Japan. To elucidate this difference, we determined the time origin of the HCV epidemic in each country by using molecularly clocked long-term serial samples obtained from HCV carriers of genotypes 1a and 1b. The molecular clock estimated that HCV genotype 1 first appeared in Japan in around 1882, whereas emergence in the U.S. was delayed until around 1910. In addition, by statistical analysis using coalescent theory, the major spread time for HCV infection in Japan occurred in the 1930s, whereas widespread dissemination of HCV in the U.S. occurred in the 1960s. These estimates of viral spread time are consistent with epidemiologic observations and predict that the burden of HCC in the U.S. will increase in the next two to three decades, possibly to equal that currently experienced in Japan.
Crystal structure of a human aminoacyl-tRNA synthetase cytokine
Authors: Yang Xiang-Lei , Skene Robert J. , McRee Duncan E. , Schimmel Paul
Published: November 2002

The 20 aminoacyl-tRNA synthetases catalyze the first step of protein synthesis and establish the rules of the genetic code through aminoacylation reactions. Biological fragments of two human enzymes, tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, connect protein synthesis to cell-signaling pathways including angiogenesis. Alternative splicing or proteolysis produces these fragments. The proangiogenic N-terminal fragment mini-TyrRS has IL-8-like cytokine activity that, like other CXC cytokines, depends on a Glu-Leu-Arg motif. Point mutations in this motif abolish cytokine activity. The full-length native TyrRS lacks cytokine activity. No structure has been available for any mammalian tRNA synthetase that, in turn, might give insight into why mini-TyrRS and not TyrRS has cytokine activities. Here, the structure of human mini-TyrRS, which contains both the catalytic and the anticodon recognition domain, is reported to a resolution of 1.18 Å. The critical Glu-Leu-Arg motif is located on an internal α-helix of the catalytic domain, where the guanidino side chain of R is part of a hydrogen-bonding network tethering the anticodon-recognition domain back to the catalytic site. Whereas the catalytic domains of the human and bacterial enzymes superimpose, the spatial disposition of the anticodon recognition domain relative to the catalytic domain is unique in mini-TyrRS relative to the bacterial orthologs. This unique orientation of the anticodon-recognition domain can explain why the fragment mini-TyrRS, and not full-length native TyrRS, is active in cytokine-signaling pathways.
Modeling the Lag Time ofListeria monocytogenesfrom Viable Count Enumeration and Optical Density Data
Authors: Baty F. , Flandrois J. P. , Delignette-Muller M. L.
Published: November 2002

The following two factors significantly influence estimates of the maximum specific growth rate (μmax) and the lag-phase duration (λ): (i) the technique used to monitor bacterial growth and (ii) the model fitted to estimate parameters. In this study, nine strains ofListeria monocytogeneswere monitored simultaneously by optical density (OD) analysis and by viable count enumeration (VCE) analysis. Four usual growth models were fitted to our data, and estimates of growth parameters were compared from one model to another and from one monitoring technique to another. Our results show that growth parameter estimates depended on the model used to fit data, whereas there were no systematic variations in the estimates of μmaxand λ when the estimates were based on OD data instead of VCE data. By studying the evolution of OD and VCE simultaneously, we found that while log OD/VCE remained constant for some of our experiments, a visible linear increase occurred during the lag phase for other experiments. We developed a global model that fits both OD and VCE data. This model enabled us to detect for some of our strains an increase in OD during the lag phase. If not taken into account, this phenomenon may lead to an underestimate of λ.
Genealogy Profiling through Strain Improvement by Using Metabolic Network Analysis: Metabolic Flux Genealogy of Several Generations of Lysine-Producing Corynebacteria
Authors: Wittmann Christoph , Heinzle Elmar
Published: November 2002

A comprehensive approach of metabolite balancing,
13
C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing
Corynebacterium glutamicum
. The five strains examined (
C. glutamicum
ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C
3
carboxylation and C
4
decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C
3
carboxylation and C
4
decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that
C. glutamicum
is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.
Macedocin, a Food-Grade Lantibiotic Produced by
Streptococcus macedonicus
ACA-DC 198
Authors: Georgalaki Marina D. , Van den Berghe Erika , Kritikos Dimitrios , Devreese Bart , Van Beeumen Jozef , Kalantzopoulos George , De Vuyst Luc , Tsakalidou Effie
Published: November 2002

Streptococcus macedonicus
ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin. Macedocin has a molecular mass of 2,794.76 ± 0.42 Da, as determined by electrospray mass spectrometry. Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen
Streptococcus pyogenes
. Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including
Clostridium tyrobutyricum
. It displays a bactericidal effect towards the most sensitive indicator strain,
Lactobacillus sakei
subsp.
sakei
LMG 13558
T
, while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production. Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121°C with 1 atm of overpressure. Inhibition of macedocin by proteolytic enzymes is variable.
Temperature- and Surfactant-Induced Membrane Modifications That Alter
Listeria monocytogenes
Nisin Sensitivity by Different Mechanisms
Authors: Li Jie , Chikindas Michael L. , Ludescher Richard D. , Montville Thomas J.
Published: November 2002

Nisin interacts with target membranes in four sequential steps: binding, insertion, aggregation, and pore formation. Alterations in membrane composition might influence any of these steps. We hypothesized that cold temperatures (10°C) and surfactant (0.1% Tween 20) in the growth medium would influence
Listeria monocytogenes
membrane lipid composition, membrane fluidity, and, as a result, sensitivity to nisin. Compared to the membranes of cells grown at 30°C, those of
L. monocytogenes
grown at 10°C had increased amounts of shorter, branched-chain fatty acids, increased fluidity (as measured by fluorescence anisotropy), and increased nisin sensitivity. When 0.1% Tween 20 was included in the medium and the cells were cultured at 30°C, there were complex changes in lipid composition. They did not influence membrane fluidity but nonetheless increased nisin sensitivity. Further investigation found that these cells had an increased ability to bind radioactively labeled nisin. This suggests that the modification of the surfactant-adapted cell membrane increased nisin sensitivity at the binding step and demonstrates that each of the four steps can contribute to nisin sensitivity.
A Novel High-Cell-Density Protein Expression System Based on
Ralstonia eutropha
Authors: Srinivasan Sriram , Barnard Gavin C. , Gerngross Tillman U.
Published: November 2002

We describe the development of a novel protein expression system based on the industrial fermentation organism
Ralstonia eutropha
(formerly known as
Alcaligenes eutrophus
) NCIMB 40124. This new system overcomes some of the shortcomings of traditional
Escherichia coli
-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in
E. coli
.
Gene Cloning and Characterization of Multiple Alkane Hydroxylase Systems inRhodococcusStrains Q15 and NRRL B-16531
Authors: Whyte L. G. , Smits T. H. M. , Labbé D. , Witholt B. , Greer C. W. , van Beilen J. B.
Published: November 2002

The alkane hydroxylase systems of twoRhodococcusstrains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1,alkB2,alkB3, andalkB4). In both strains, thealkB1andalkB2homologs were part ofalkgene clusters, each encoding two rubredoxins (rubA1andrubA2;rubA3andrubA4), a putative TetR transcriptional regulatory protein (alkU1;alkU2), and, in thealkB1cluster, a rubredoxin reductase (rubB). ThealkB3andalkB4homologs were found as separate genes which were not part ofalkgene clusters. Functional heterologous expression of some of the rhodococcalalkgenes (alkB2,rubA2, andrubA4[NRRL B-16531];alkB2andrubB[Q15]) was achieved inEscherichia coliandPseudomonasexpression systems.Pseudomonasrecombinants containing rhodococcalalkB2were able to mineralize and grow on C12to C16n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to thePseudomonas putidaGPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported inRhodococcus.
Arthrobacter aurescens
TC1 Metabolizes Diverse
s
-Triazine Ring Compounds
Authors: Strong Lisa C. , Rosendahl Charlotte , Johnson Gilbert , Sadowsky Michael J. , Wackett Lawrence P.
Published: November 2002

Arthrobacter aurescens
strain TC1 was isolated without enrichment by plating atrazine-contaminated soil directly onto atrazine-clearing plates.
A. aurescens
TC1 grew in liquid medium with atrazine as the sole source of nitrogen, carbon, and energy, consuming up to 3,000 mg of atrazine per liter.
A. aurescens
TC1 is metabolically diverse and grew on a wider range of
s
-triazine compounds than any bacterium previously characterized. The 23
s
-triazine substrates serving as the sole nitrogen source included the herbicides ametryn, atratone, cyanazine, prometryn, and simazine. Moreover, atrazine substrate analogs containing fluorine, mercaptan, and cyano groups in place of the chlorine substituent were also growth substrates. Analogs containing hydrogen, azido, and amino functionalities in place of chlorine were not growth substrates.
A. aurescens
TC1 also metabolized compounds containing chlorine plus
N
-ethyl,
N
-propyl,
N
-butyl,
N
-
s
-butyl,
N
-isobutyl, or
N
-
t
-butyl substituents on the
s
-triazine ring. Atrazine was metabolized to alkylamines and cyanuric acid, the latter accumulating stoichiometrically. Ethylamine and isopropylamine each served as the source of carbon and nitrogen for growth. PCR experiments identified genes with high sequence identity to
atzB
and
atzC
, but not to
atzA
, from
Pseudomonas
sp. strain ADP.
Oligonucleotide Fingerprinting of rRNA Genes for Analysis of Fungal Community Composition
Authors: Valinsky Lea , Della Vedova Gianluca , Jiang Tao , Borneman James
Published: November 2002

Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones derived from soil generated 455 clusters. A pairwise sequence analysis showed that clones with average sequence identities of 99.2% were grouped into the same cluster. To examine the accuracy of the taxonomic identities produced by this OFRG experiment, we determined the nucleotide sequences for 117 clones distributed throughout the tree. For all but two of these clones, the taxonomic identities generated by this OFRG experiment were consistent with those generated by a nucleotide sequence analysis. Eighty-eight percent of the clones were affiliated withAscomycota, while 12% belonged toBasidiomycota. A large fraction of the clones were affiliated with the generaFusarium(404 clones) andRaciborskiomyces(176 clones). Smaller assemblages of clones had high sequence identities to theAlternaria,Ascobolus,Chaetomium,Cryptococcus, andRhizoctoniaclades.
Bile Stress Response inListeria monocytogenesLO28: Adaptation, Cross-Protection, and Identification of Genetic Loci Involved in Bile Resistance
Authors: Begley Máire , Gahan Cormac G. M. , Hill Colin
Published: November 2002

Bile is one of many barriers thatListeria monocytogenesmust overcome in the human gastrointestinal tract in order to infect and cause disease. We demonstrated that stationary-phase cultures ofL. monocytogenesLO28 were able to tolerate concentrations of bovine, porcine, and human bile and bile acids well in excess of those encountered in vivo. Strain LO28 was relatively bile resistant compared with other clinical isolates ofL. monocytogenes, as well as withListeria innocua,Salmonella entericaserovar Typhimurium LT2, andLactobacillus sakei. While exponential-phaseL. monocytogenesLO28 cells were exquisitely sensitive to unconjugated bile acids, prior adaptation to sublethal levels of bile acids or heterologous stresses, such as acid, heat, salt, or sodium dodecyl sulfate (SDS), significantly enhanced bile resistance. This adaptive response was independent of protein synthesis, and in the cases of bile and SDS adaptation, occurred in seconds. In order to identify genetic loci involved in the bile tolerance phenotype ofL. monocytogenesLO28, transposon (Tn917) and plasmid (pORI19) integration banks were screened for bile-sensitive mutants. The disrupted genes included a homologue of thecapAlocus required for capsule formation inBacillus anthracis; a gene encoding the transcriptional regulator ZurR; a homologue of anEscherichia coligene,lytB, involved in isoprenoid biosynthesis; a gene encoding a homologue of theBacillus subtilismembrane protein YxiO; and a gene encoding an amino acid transporter with a putative role in pH homeostasis,gadE. Interestingly, all of the identified loci play putative roles in maintenance of the cell envelope or in stress responses.
Photoreactivation of
Escherichia coli
after Low- or Medium-Pressure UV Disinfection Determined by an Endonuclease Sensitive Site Assay
Authors: Oguma Kumiko , Katayama Hiroyuki , Ohgaki Shinichiro
Published: November 2002

Photoreactivation of
Escherichia coli
after inactivation by a low-pressure (LP) UV lamp (254 nm), by a medium-pressure (MP) UV lamp (220 to 580 nm), or by a filtered medium-pressure (MPF) UV lamp (300 to 580 nm) was investigated. An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genomic DNA of
E. coli
, while a conventional cultivation assay was used to investigate the colony-forming ability (CFA) of
E. coli.
In photoreactivation experiments, more than 80% of the pyrimidine dimers induced by LP or MPF UV irradiation were repaired, while almost no repair of dimers was observed after MP UV exposure. The CFA ratios of
E. coli
recovered so that they were equivalent to 0.9-, 2.3-, and 1.7-log inactivation after 3-log inactivation by LP, MP, and MPF UV irradiation, respectively. Photorepair treatment of DNA in vitro suggested that among the MP UV emissions, wavelengths of 220 to 300 nm reduced the subsequent photorepair of ESS, possibly by causing a disorder in endogenous photolyase, an enzyme specific for photoreactivation. On the other hand, the MP UV irradiation at wavelengths between 300 and 580 nm was observed to play an important role in reducing the subsequent recovery of CFA by inducing damage other than damage to pyrimidine dimers. Therefore, it was found that inactivating light at a broad range of wavelengths effectively reduced subsequent photoreactivation, which could be an advantage that MP UV irradiation has over conventional LP UV irradiation.
Impact of Integrated Fish Farming on Antimicrobial Resistance in a Pond Environment
Authors: Petersen Andreas , Andersen Jens Strodl , Kaewmak Tawatchai , Somsiri Temdoung , Dalsgaard Anders
Published: November 2002

Integrated fish farming combines livestock production with fish farming. Animal manure is shed directly into a fish pond as fertilizer and supports the growth of photosynthetic organisms. The livestock, mainly chickens and pigs, is often fed feed containing growth promoters. In this study we investigated the impact of integrated fish farming on the levels of antimicrobial-resistant bacteria in a pond environment. One integrated broiler chicken-fish farm was studied for 2 months immediately after the start of a new fish production cycle. A significant increase over time in the resistance to six different antimicrobials was found for the indicator organism
Acinetobacter
spp. isolated from composite water-sediment samples. The initial resistance levels prior to the new production cycle were 1 to 5%. After 2 months the levels of resistance to oxytetracycline and sulfamethoxazole reached 100%, and the levels of resistance to ciprofloxacin were more than 80%. The long-term effects of resistance on integrated farming were studied on seven additional farms. The resistance levels were particularly high among
Enterococcus
spp. and were also high among
Acinetobacter
spp. isolated from water-sediment samples compared to the resistance levels at four control farms. In conclusion, integrated fish farming seems to favor antimicrobial-resistant bacteria in the pond environment. This could be attributed to the selective pressure of antimicrobials in the pond environment and/or to the introduction of antimicrobial-resistant bacteria from animal manure. Potential risks to human health were not addressed in this study and remain to be elucidated.
The Product of
arcR
, the Sixth Gene of the
arc
Operon of
Lactobacillus sakei
, Is Essential for Expression of the Arginine Deiminase Pathway
Authors: Zúñiga Manuel , Miralles María del Carmen , Pérez-Martínez Gaspar
Published: November 2002

Lactobacillus sakei
is a lactic acid bacterium commonly used as a starter culture for dry sausage production and can utilize arginine via the arginine deiminase pathway. The
arcABCTD
cluster of
L. sakei
has been characterized, and transcriptional studies have shown that its expression is subject to carbon catabolite repression and induction by arginine. Downstream of
arcD
an additional gene has been found; this gene,
arcR
, codes for a putative regulatory protein of the Crp/Fnr family. Transcriptional studies have shown that
arcR
is coordinately transcribed with the remaining
arc
genes, and therefore, these genes constitute the
arcABCTDR
operon. Northern analysis also showed a complex pattern of transcripts, suggesting that processing and partial termination may play a role in regulation of the expression of individual genes of the operon. Inactivation of
arcR
led to arrest of transcription of the operon, indicating that the ArcR protein is essential for expression of the
arc
genes. The availability of this mutant made it possible to study whether the ability to utilize arginine affects the growth of
L. sakei
in meat fermentations. Under our experimental conditions, expression of arginine deiminase does not confer an obvious advantage to
L. sakei
, since the wild type and an
arcR
mutant strain displayed similar dynamics of growth.