bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhancement of lipopolysaccharide‐stimulated JNK activity in rat aortic smooth muscle cells by pharmacological and adenovirus‐mediated inhibition of inhibitory kappa B kinase signalling
Authors: MacKenzie Christopher J , Paul Andrew , Wilson Susan , de Martin Rainer , Baker Andrew H , Plevin Robin 
Published: July 2003

In rat aortic smooth muscle cells (RASMCs), the putative nuclear factor kappa B (NFκB) inhibitor Pyrrolidine dithiocarbamate (PDTC) was found to inhibit lipopolysaccharide (LPS)‐stimulated NFκB DNA‐binding. However, further investigation identified the site of inhibition as being at, or upstream of, the inhibitory kappa B kinases (IKKs) as their kinase activity was substantially reduced.
In addition, PDTC potentiated LPS‐stimulated c‐Jun N‐terminal kinase (JNK), p38 mitogen‐activated protein kinase (MAP kinase) and MAP kinase‐activated protein kinase‐2 activity (the downstream target of p38 MAP kinase).
Another inhibitor of NFκB signalling, the serine protease inhibitor Nαp‐tosyl‐L‐lysine chloro‐methylketone (TLCK), also inhibited LPS‐stimulated IKK activity and potentiated JNK activity in response to LPS, suggesting that cross‐talk may occur between the NFκB and stress‐activated protein kinase pathways at the level of IKK or at a common point upstream.
Infection of RASMCs with an adenovirus encoding either inhibitory kappa Bα or a dominant‐negative IKKβ potentiated LPS‐stimulated JNK activity.
These studies therefore suggest that the loss of NFκB DNA‐binding and resultant transcriptional activity, rather than the loss of IKK activity, is sufficient to cause an increase in JNK activity. This shows that either pharmacological or molecular inhibition of NFκB DNA‐binding enhances JNK activation in vascular smooth muscle cells, an effect that may contribute to the pathophysiological effects of LPS.

British Journal of Pharmacology (2003) 139, 1041–1049. doi:10.1038/sj.bjp.0705330
Vitrification of cryoelectron microscopy specimens revealed by high‐speed photographic imaging
Authors: Kasas S. , Dumas G. , Dietler G. , Catsicas S. , Adrian M. 
Published: July 2003

SummaryCryoelectron microsopy is a widely used technique to observe biological material in an almost physiological, fully hydrated state. The sample is prepared for electron microsopy observation by quickly reducing its temperature to −180 °C. The high‐speed cooling induces the formation of vitreous water, which preserves the sample conformation. However, the way vitrification occurs is still poorly understood. In order to better understand the phenomenon, we have used a stroboscopic device to visualize the interaction between the electron microscopy grid and the cryogen. By blocking the free fall of the plunger once the grid has penetrated the coolant by half its diameter, we have elucidated the way in which vitrification propagates. The findings were confirmed by numerical simulation. In addition, according to our observations, we now present an alternative way to prepare vitreous specimens. This new method, with the grid parallel to the liquid cryogen surface, decreases evaporation from the sample during its free fall towards the coolant and at the same time achieves a more uniform vitrification over the entire surface of the specimen.
Attenuation correction in confocal laser microscopes: a novel two‐view approach
Authors: Can A. , Al‐Kofahi O. , Lasek S. , Szarowski D. H. , Turner J. N. , Roysam B. 
Published: July 2003

SummaryConfocal microscopy is a three‐dimensional (3D) imaging modality, but the specimen thickness that can be imaged is limited by depth‐dependent signal attenuation. Both software and hardware methods have been used to correct the attenuation in reconstructed images, but previous methods do not increase the image signal‐to‐noise ratio (SNR) using conventional specimen preparation and imaging. We present a practical two‐view method that increases the overall imaging depth, corrects signal attenuation and improves the SNR. This is achieved by a combination of slightly modified but conventional specimen preparation, image registration, montage synthesis and signal reconstruction methods. The specimen is mounted in a symmetrical manner between a pair of cover slips, rather than between a slide and a cover slip. It is imaged sequentially from both sides to generate two 3D image stacks from perspectives separated by approximately 180° with respect to the optical axis. An automated image registration algorithm performs a precise 3D alignment, and a model‐based minimum mean squared algorithm synthesizes a montage, combining the content of both the 3D views. Experiments with images of individual neurones contrasted with a space‐filling fluorescent dye in thick brain tissue slices produced precise 3D montages that are corrected for depth‐dependent signal attenuation. The SNR of the reconstructed image is maximized by the method, and it is significantly higher than in the single views after applying our attenuation model. We also compare our method with simpler two‐view reconstruction methods and quantify the SNR improvement. The reconstructed images are a more faithful qualitative visualization of the specimen's structure and are quantitatively more accurate, providing a more rigorous basis for automated image analysis.
Algorithms for accurate 3D registration of neuronal images acquired by confocal scanning laser microscopy
Authors: Al‐Kofahi O. , Can A. , Lasek S. , Szarowski D. H. , Turner J. N. , Roysam B. 
Published: July 2003

SummaryThis paper presents automated and accurate algorithms based on high‐order transformation models for registering three‐dimensional (3D) confocal images of dye‐injected neurons. The algorithms improve upon prior methods in several ways, and meet the more stringent image registration needs of applications such as two‐view attenuation correction recently developed by us. First, they achieve high accuracy (≈ 1.2 voxels, equivalent to 0.4 µm) by using landmarks, rather than intensity correlations, and by using a high‐dimensional affine and quadratic transformation model that accounts for 3D translation, rotation, non‐isotropic scaling, modest curvature of field, distortions and mechanical inconsistencies introduced by the imaging system. Second, they use a hierarchy of models and iterative algorithms to eliminate potential instabilities. Third, they incorporate robust statistical methods to achieve accurate registration in the face of inaccurate and missing landmarks. Fourth, they are fully automated, even estimating the initial registration from the extracted landmarks. Finally, they are computationally efficient, taking less than a minute on a 900‐MHz Pentium III computer for registering two images roughly 70 MB in size. The registration errors represent a combination of modelling, estimation, discretization and neuron tracing errors. Accurate 3D montaging is described; the algorithms have broader applicability to images of vasculature, and other structures with distinctive point, line and surface landmarks.
Time‐gated total internal reflection fluorescence spectroscopy (TG‐TIRFS): application to the membrane marker laurdan
Authors: Schneckenburger H. , Stock K. , Strauss W. S. L. , Eickholz J. , Sailer R. 
Published: July 2003

SummaryA novel setup for total internal reflection fluorescence microscopy with spectral and temporal (nanosecond) resolution was used to measure the emission spectra of the membrane marker laurdan either selectively within the plasma membrane or in whole living cells, depending on the incident angle of the excitation light. With increasing temperature, the intensity of the fluorescence band around 490 nm increased in comparison with the band around 440 nm, which has previously been assigned to a phase transition of membrane lipids from gel to liquid crystalline phase. For a better separation of the overlapping spectral bands, time‐gated detection with a delay of 10–15 ns with respect to the exciting laser pulse was used. As a parameter of membrane dynamics the so‐called generalized polarization GP = (I440 − I490)/(I440 + I490) was evaluated at temperatures between 24 and 41 °C and variable angles of the incident light permitting to excite laurdan molecules either within the plasma membrane or in the whole cell. A decrease of the GP values by ≈ 0.2 units between 28 and 41 °C indicated an increase in membrane fluidity or a decrease in membrane stiffness with increasing temperature. In addition, higher GP values were observed for the plasma membrane as compared with intracellular membranes, probably due to a higher amount of cholesterol. Because properties of the plasma membrane have a large influence on the uptake or release of certain pharmaceutical agents or metabolites, the direct assessment of the dynamics of the plasma membrane by total internal reflection fluorescence spectroscopy appears to be important for pharmacology.
Semi‐automated software for the three‐dimensional delineation of complex vascular networks
Authors: Barber P. R. , Vojnovic B. , Ameer‐Beg S. M. , Hodgkiss R. J. , Tozer G. M. , Wilson J. 
Published: July 2003

SummaryThe understanding of tumour angiogenesis is of great importance in cancer research, as is the tumour response to vascular‐targeted drugs. This paper presents software aimed at aiding these investigations and other situations where linear or dendritic structures are to be delineated from three‐dimensional (3D) data sets. This software application was written to analyse the data from 3D data sets by allowing the manual and semi‐automated tracking and delineation of the vascular tree, including the measurement of vessel diameter. A new algorithm, CHARM, based on a compact Hough transform and the formation of a radial map, has been used to locate vessel centres and measure diameters automatically. The robustness of this algorithm to image smoothing and noise has been investigated.
Inhibition of protein adsorption to muscovite mica by monovalent cations
Authors: Czajkowsky D. M. , Shao Z. 
Published: July 2003

SummaryOne of the most challenging steps in biological atomic force microscopy (AFM) is to find conditions under which the sample will adsorb to a substrate. Here we show that a common constituent of biological buffers, monovalent cations, can inhibit the adsorption of a number of different proteins onto one of the best substrates for biological AFM, muscovite mica. The potency series for different cations to prevent adsorption is the same for every protein, K+ > Na+ > Li+, and, in each case, this inhibition could be overcome by increasing the concentration of proteins. These results thus suggest that reducing the extent of this inhibition by using lower concentrations of salt, higher concentrations of proteins, or Li+ in place of K+ and Na+ may be generally useful procedures to maximize the amount of protein on mica.
Enzymuria following amphotericin B application in the rat
Authors: Inselmann Gerhard , Balaschke Michael , Heidemann Hugo T. 
Published: July 2003

SummaryThe effect of amphotericin B application on urinary renal tubule enzyme excretion was investigated in rats treated with amphotericin B (1.5 mg kg−1 b.i.d., i.v.) for 4 days. Application of amphotericin B induced a significant higher daily urinary enzyme activity of the renal tubular enzymes N‐acetyl‐β‐glucosaminidase (NAG), β‐glucuronidase (GRS), alanine‐aminopeptidase (AAP) and γ‐glutamyltransferase (GGT) in comparison with controls and sodium deoxycholate treated animals as well. A significant increase in the renal excretion of NAG, GRS, AAP and GGT occurred after the first day of amphotericin B treatment and continued until the fourth day. Following treatment for 4 days with amphotericin B urine AAP activity amounted to 69 ± 19 U g−1 creatinine, control: 39 ± 7 U g−1 creatinine (P < 0.05). After 4 days GGT excretion increased to 803 ± 238 U g−1 creatinine, control: 445 ± 106 U g−1 creatinine (P < 0.05). At the fourth day NAG excretion was 80 ± 39 U g−1 creatinine, control: 23 ± 5 U g−1 creatinine (P < 0.05) and GRS 724 ± 604 U g−1 creatinine (amphotericin B), control: 276 ± 158 U g−1 creatinine (P < 0.05). Treatment with amphotericin B decreased the creatinine clearance significantly: 0.94 ± 0.16 ml−1 min−1 vs. control 1.35 ± 0.29 ml−1min−1 (P < 0.05). Fractional sodium and potassium excretion was not influenced by amphotericin B. The application of sodium deoxycholate had no influence on urinary renal tubular enzyme activity. The results show that amphotericin B application induces early enzymuria of renal tubule enzymes suggesting damage of proximal renal tubules.
Paracoccidioidomycosis and larynx carcinoma
Authors: Maymó Argañaraz M. , Luque A. G. , Tosello M. E. A. , Perez J. 
Published: July 2003

SummaryReport of a case of paracoccidioidomycosis associated with a carcinoma: both located in the larynx in a patient whose therapeutic response to antifungal treatment produced a recovery of physical conditions. This case shows the importance of taking into account the diagnosis of paracoccidioidomycosis in all patients with problems in the larynx, especially those who inhabit or inhabited endemic areas of Paracoccidioides brasiliensis.
Isolation of dermatophytes and other keratinophilic fungi from soils of Jammu, India
Authors: Deshmukh S. K. , Agrawal S. C. 
Published: July 2003

SummaryA total of 112 soil samples collected from various areas of Jammu, India, were screened for the prevalence of keratinophilic fungi and related dermatophytes. From 65 positive samples (58.1%), a total of six genera with 13 species were isolated.
Purification and Characterization of a Novel Erythrose Reductase from
Candida magnoliae
Authors: Lee Jung-Kul , Kim Sang-Yong , Ryu Yeon-Woo , Seo Jin-Ho , Kim Jung-Hoe 
Published: July 2003

ABSTRACT

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from
Candida magnoliae
KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases.
C. magnoliae
erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (
K

m

= 7.9 mM;
k
cat
/
K

m

= 0.73 mM
−1
s
−1
). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (
k
cat
/
K

m

= 450 mM
−1
s
−1
) than with NADPH (
k
cat
/
K

m

= 5.5 mM
−1
s
−1
), unlike previously characterized aldose reductases, and is specific for transferring the 4-
pro-R
hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu
2+
and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the
C. magnoliae
erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.
Characterization and In Situ Carbon Metabolism of Phototrophic Consortia
Authors: Glaeser Jens , Overmann Jörg 
Published: July 2003

ABSTRACT

A dense population of the phototrophic consortium “
Pelochromatium roseum
” was investigated in the chemocline of a temperate holomictic lake (Lake Dagow, Brandenburg, Germany). Fluorescence in situ hybridization revealed that the brown epibionts of “
P. roseum
” constituted up to 37% of the total bacterial cell number and up to 88% of all green sulfur bacteria present in the chemocline. Specific amplification of 16S rRNA gene fragments of green sulfur bacteria and denaturing gradient gel electrophoresis fingerprinting yielded a maximum of four different DNA bands depending on the year of study, indicating that the diversity of green sulfur bacteria was low. The 465-bp 16S rRNA gene sequence of the epibiont of “
P. roseum
” was obtained after sorting of individual consortia by micromanipulation, followed by a highly sensitive PCR. The sequence obtained represents a new phylotype within the radiation of green sulfur bacteria. Maximum light-dependent H
14
CO
3
−
fixation in the chemocline in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that there was anaerobic autotrophic growth of the green sulfur bacteria. The metabolism of the epibionts was further studied by determining stable carbon isotope ratios (δ
13
C) of their specific biomarkers. Analysis of photosynthetic pigments by high-performance liquid chromatography revealed the presence of high concentrations of bacteriochlorophyll (BChl)
e
and smaller amounts of BChl
a
and
d
and chlorophyll
a
in the chemocline. Unexpectedly, isorenieratene and β-isorenieratene, carotenoids typical of other brown members of the green sulfur bacteria, were absent. Instead, four different esterifying alcohols of BChl
e
were isolated as biomarkers of green sulfur bacterial epibionts, and their δ
13
C values were determined. Farnesol, tetradecanol, hexadecanol, and hexadecenol all were significantly enriched in
13
C compared to bulk dissolved and particulate organic carbon and compared to the biomarkers of purple sulfur bacteria. The difference between the δ
13
C values of farnesol, the major esterifying alcohol of BChl
e
, and CO
2
was −7.1%, which provides clear evidence that the mode of growth of the green sulfur bacterial epibionts of “
P. roseum
” in situ is photoautotrophic.
Dynamics of Fungal Communities in Bulk and Maize Rhizosphere Soil in the Tropics
Authors: Gomes Newton C. Marcial , Fagbola Olajire , Costa Rodrigo , Rumjanek Norma Gouvea , Buchner Arno , Mendona-Hagler Leda , Smalla Kornelia 
Published: July 2003

ABSTRACT

The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data. Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years. A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing. A rhizosphere effect was observed for fungal populations at all stages of plant development. In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE. Similar types of fingerprints were observed in two consecutive growth periods. No major differences were detected in the fungal patterns of the two cultivars. Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands. The clones were characterized by their
Hin
fI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced. The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands. Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.
Effects of Litter Addition on Ectomycorrhizal Associates of a Lodgepole Pine (
Pinus contorta
) Stand in Yellowstone National Park
Authors: Cullings Kenneth W. , New Michael H. , Makhija Shilpa , Parker V. Thomas 
Published: July 2003

ABSTRACT

Increasing soil nutrients through litter manipulation, pollution, or fertilization can adversely affect ectomycorrhizal (EM) communities by inhibiting fungal growth. In this study, we used molecular genetic methods to determine the effects of litter addition on the EM community of a
Pinus contorta
stand in Yellowstone National Park that regenerated after a stand-replacing fire. Two controls were used; in unmodified control plots nothing was added to the soil, and in perlite plots perlite, a chemically neutral substance, was added to maintain soil moisture and temperature at levels similar to those under litter. We found that (i) species richness did not change significantly following perlite addition (2.6 ± 0.3 species/core in control plots, compared with 2.3 ± 0.3 species/core in perlite plots) but decreased significantly (
P
< 0.05) following litter addition (1.8 ± 0.3 species/core); (ii) EM infection was not affected by the addition of perlite but increased significantly (
P
< 0.001) in response to litter addition, and the increase occurred only in the upper soil layer, directly adjacent to the added litter; and (iii)
Suillus granulatus
,
Wilcoxina mikolae
, and agaricoid DD were the dominant organisms in controls, but the levels of
W. mikolae
and agaricoid DD decreased significantly in response to both perlite and litter addition. The relative levels of
S. granulatus
and a fourth fungus, Cortinariaceae species 2, increased significantly (
P
< 0.01 and
P
< 0.05, respectively) following litter addition. Thus, litter addition resulted in some negative effects that may be attributable to moisture-temperature relationships rather than to the increased nutrients associated with litter. Some species respond positively to litter addition, indicating that there are differences in their physiologies. Hence, changes in the EM community induced by litter accumulation also may affect ecosystem function.
Construction of an Expression System for Site-DirectedMutagenesis of the LantibioticMersacidin
Authors: Szekat Christiane , Jack Ralph W. , Skutlarek Dirk , Färber Harald , Bierbaum Gabriele 
Published: July 2003

ABSTRACT

The
lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an
antimicrobial peptide of 20 amino acids which is produced by
Bacillus
sp. strain HIL Y-85,54728. Mersacidin inhibits
bacterial cell wall biosynthesis by binding to the precursor molecule
lipid II. The structural gene of mersacidin (
mrsA
) and the
genes for the enzymes of the biosynthesis pathway, dedicated
transporters, producer self-protection proteins, and regulatory factors
are organized in a biosynthetic gene cluster. For
site-directed mutagenesis of lantibiotics, the engineered genes must be
expressed in an expression system that contains all of the factors
necessary for biosynthesis, export, and producer self-protection. In
order to express engineered mersacidin peptides, a system in which the
engineered gene replaces the wild-type gene on the chromosome was
constructed. To test the expression system, three mutants were
constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at
position 16 was replaced with the Ile residue found in the closely
related lantibiotic actagardine. S16I mersacidin was produced only in
small amounts. The purified peptide had markedly reduced antimicrobial
activity, indicating an essential role for Dha16 in biosynthesis and
biological activity of mersacidin. Similarly, Glu17, which is thought
to be an essential structure in mersacidin, was exchanged for alanine.
E17A mersacidin was obtained in good yields but also showed markedly
reduced activity, thus confirming the importance of the carboxylic acid
function at position 17 in the biological activity of mersacidin.
Finally, the exchange of an aromatic for an aliphatic hydrophobic
residue at position 3 resulted in the mutant peptide F3L mersacidin;
this peptide showed only moderately reduced
activity.