bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and Conformational Basis of a Specific and High-Affinity Interaction between AlbA and Albicidin Phytotoxin
Authors: Weng Li-Xing , Wang Lian-Hui , Xu Jin-Ling , Wu Ji-En , Li Qi , Zhang Lian-Hui
Published: March 2005

The
albA
gene of
Klebsiella oxytoca
encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 × 10
−8
M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys
106
, Trp
110
, Tyr
113
, Leu
114
, Tyr
126
, Pro
134
, and Trp
162
with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His
125
, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.
Functional Characterization of Two Cytochrome P450 Monooxygenase Genes,
P450
-
1
and
P450
-
4
, of the Gibberellic Acid Gene Cluster in
Fusarium proliferatum
(
Gibberella fujikuroi
MP-D)
Authors: Malonek S. , Rojas M. C. , Hedden P. , Gaskin P. , Hopkins P. , Tudzynski B.
Published: March 2005

Gibberella fujikuroi
is a species complex with at least nine different biological species, termed mating populations (MPs) A to I (MP-A to MP-I), known to produce many different secondary metabolites. So far, gibberellin (GA) production is restricted to
Fusarium fujikuroi
(
G. fujikuroi
MP-C), although at least five other MPs contain all biosynthetic genes. Here, we analyze the GA gene cluster and GA pathway in the closest related species,
Fusarium proliferatum
(MP-D), and demonstrate that the GA genes share a high degree of sequence homology with the corresponding genes of MP-C. The GA production capacity was restored after integration of the entire GA gene cluster from MP-C, indicating the existence of an active regulation system in
F. proliferatum
. The results further indicate that one reason for the loss of GA production is the accumulation of several mutations in the coding and 5′ noncoding regions of the
ent
-kaurene oxidase gene,
P450
-
4
.
“
Endomicrobia
”: Cytoplasmic Symbionts of Termite Gut Protozoa Form a Separate Phylum of Prokaryotes
Authors: Stingl Ulrich , Radek Renate , Yang Hong , Brune Andreas
Published: March 2005

Lignocellulose digestion by wood-feeding termites depends on the mutualistic interaction of unusual, flagellate protists located in their hindgut. Most of the flagellates harbor numerous prokaryotic endosymbionts of so-far-unknown identity and function. Using a full-cycle molecular approach, we show here that the endosymbionts of the larger gut flagellates of
Reticulitermes santonensis
belong to the so-called termite group 1 (TG-1) bacteria, a group of clones previously obtained exclusively from gut homogenates of
Reticulitermes speratus
that are only distantly related to other bacteria and are considered a novel bacterial phylum based on their 16S rRNA gene sequences. Fluorescence in situ hybridization with specifically designed oligonucleotide probes confirmed that TG-1 bacteria are indeed located within the flagellate cells and demonstrated that
Trichonympha agilis
(Hypermastigida) and
Pyrsonympha vertens
(Oxymonadida) harbor phylogenetically distinct populations of symbionts (<95% sequence similarity). Transmission electron microscopy revealed that the symbionts are small, spindle-shaped cells (0.6 μm in length and 0.3 μm in diameter) surrounded by two membranes and located within the cytoplasm of their hosts. The symbionts of the two flagellates are described as candidate species in the candidate genus “
Endomicrobium
.” Moreover, we provide evidence that the members of the TG-1 phylum, for which we propose the candidate name “
Endomicrobia
,” are phylogenetically extremely diverse and are present in and also restricted to the guts of all lower termites and wood-feeding cockroaches of the genus
Cryptocercus
, the only insects that are in an exclusive, obligately mutualistic association with such unique cellulose-fermenting protists.
Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms
Authors: Scheldeman Patsy , Pil Annelies , Herman Lieve , De Vos Paul , Heyndrickx Marc
Published: March 2005

The presence of highly heat-resistant spores ofBacillus sporothermoduransin ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100°C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera.Bacillus licheniformisandBacillus palliduswere the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.
Overproduction of Heterologous Mannitol 1-Phosphatase: a Key Factor for Engineering Mannitol Production byLactococcus lactis
Authors: Wisselink H. Wouter , Moers Antoine P. H. A. , Mars Astrid E. , Hoefnagel Marcel H. N. , de Vos Willem M. , Hugenholtz Jeroen
Published: March 2005

To achieve high mannitol production byLactococcus lactis, the mannitol 1-phosphatase gene ofEimeria tenellaand the mannitol 1-phosphate dehydrogenase genemtlDofLactobacillus plantarumwere cloned in the nisin-dependentL. lactisNICE overexpression system. As predicted by a kineticL. lactisglycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by theL. lactisparental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficientL. lactisstrain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.
Intracellular pH Distribution in
Saccharomyces cerevisiae
Cell Populations, Analyzed by Flow Cytometry
Authors: Valli Minoska , Sauer Michael , Branduardi Paola , Borth Nicole , Porro Danilo , Mattanovich Diethard
Published: March 2005

Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of
Saccharomyces cerevisiae
cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on
S. cerevisiae
cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.
Two α(1-3) Glucan Synthases with Different Functions in
Aspergillus fumigatus
Authors: Beauvais A. , Maubon D. , Park S. , Morelle W. , Tanguy M. , Huerre M. , Perlin D. S. , Latgé J. P.
Published: March 2005

α(1-3) glucan is a main component of the
Aspergillus fumigatus
cell wall. In spite of its importance, synthesis of this amorphous polymer has not been investigated to date. Two genes in
A. fumigatus
,
AGS1
and
AGS2
, are highly homologous to the
AGS
genes of
Schizosaccharomyces pombe
, which encode putative α(1-3) glucan synthases. The predicted Ags proteins of
A. fumigatus
have an estimated molecular mass of 270 kDa.
AGS1
and
AGS2
were disrupted in
A. fumigatus
. Both Δags mutants have similar altered hyphal morphologies and reduced conidiation levels. Only Δags1 presented a reduction in the α(1-3) glucan content of the cell wall. These results showed that Ags1p and Ags2p were functionally different. The cellular localization of the two proteins was in agreement with their different functions: Ags1p was localized at the periphery of the cell in connection with the cell wall, whereas Ags2p was intracellularly located. An original experimental model of invasive aspergillosis based on mixed infection and quantitative PCR was developed to analyze the virulence of
A. fumigatus
mutant and wild-type strains. Using this model, it was shown that the cell wall and morphogenesis defects of Δags1 and Δags2 were not associated with a reduction in virulence in either mutant. This result showed that a 50% reduction in the content of the cell wall α(1-3) glucan does not play a significant role in
A. fumigatus
pathogenicity.
Role of
cis
-Acting Sites
NorL
, a TATA Box, and
AflR1
in
nor-1
Transcriptional Activation in
Aspergillus parasiticus
Authors: Miller Michael J. , Roze Ludmila V. , Trail Frances , Linz John E.
Published: March 2005

The transcription factor AflR is required for up-regulation of specific pathway genes involved in aflatoxin biosynthesis in the filamentous fungus
Aspergillus. nor-1
encodes an early aflatoxin pathway enzyme; its promoter contains a consensus AflR binding site (
AflR1
). Proteins in
Aspergillus parasiticus
cell extracts and AflR expressed in
Escherichia coli
do not bind to
A. parasiticus AflR1
in vitro, so it was not clear if this site was required for
nor-1
expression or if other transcription factors contributed to gene regulation. In this study we defined the role of AflR1 in
nor-1
expression in
A. parasiticus
and identified additional
cis
-acting sites required for maximum
nor-1
transcriptional activation. Deletion and substitution of
AflR1
in the
nor-1
promoter in
A. parasiticus nor-1
::GUS reporter strains showed that this site is required for
nor-1
transcriptional activation in vivo. Substitution of a putative TATA box in the
nor-1
promoter resulted in nondetectable β-glucuronidase (GUS) activity, demonstrating that this TATA box is functional in vivo. We also identified a novel
cis
-acting site, designated
NorL
, between residues −210 and −238 that was required for maximum
nor-1
transcriptional activation in
A. parasiticus
grown in liquid medium and on solid medium. Using an electrophoretic mobility shift assay, we identified a specific
NorL
-dependent DNA-protein complex that relies on a functional AflR, either directly or indirectly, for maximum binding capacity. Because the
NorL
site appears only once in the aflatoxin gene cluster, its association with the
nor-1
promoter may have important implications for the overall regulatory scheme for the aflatoxin pathway.
Coexistence of Bacterial Sulfide Oxidizers, Sulfate Reducers, and Spirochetes in a Gutless Worm (Oligochaeta) from the Peru Margin
Authors: Blazejak Anna , Erséus Christer , Amann Rudolf , Dubilier Nicole
Published: March 2005

Olavius crassitunicatusis a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distantO. crassitunicatuspopulations (separated by 385 km) on the Peru margin (water depth, ∼300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the γ-Proteobacteria(Gamma 1 and 2 symbionts), two members of the δ-Proteobacteria(Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the δ-Proteobacteria(Delta 3 symbiont) was found in only one of the two host populations. Three of theO. crassitunicatusbacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts ofOlavius algarvensis,Olavius loisae, andInanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts ofO. algarvensis, and the spirochete is closely related to spirochetal symbionts ofO. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.
Natural Dissemination of
Bacillus anthracis
Spores in Northern Canada
Authors: Dragon D. C. , Bader D. E. , Mitchell J. , Woollen N.
Published: March 2005

Soil samples were collected from around fresh and year-old bison carcasses and areas not associated with known carcasses in Wood Buffalo National Park during an active anthrax outbreak in the summer of 2001. Sample selection with a grid provided the most complete coverage of a site. Soil samples were screened for viable
Bacillus anthracis
spores via selective culture, phenotypic analysis, and PCR.
Bacillus anthracis
spores were isolated from 28.4% of the samples. The highest concentrations of
B. anthracis
spores were found directly adjacent to fresh carcasses and invariably corresponded to locations where the soil had been saturated with body fluids escaping the carcass through either natural body orifices or holes torn by scavengers. The majority of positive samples were found within 2 m of both year-old and fresh carcasses and probably originated from scavengers churning up and spreading the body fluid-saturated soil as they fed. Trails of lesser contamination radiating from the carcasses probably resulted from spore dissemination through adhesion to scavengers and through larger scavengers dragging away disarticulated limbs. Comparison of samples from minimally scavenged and fully necropsied carcass sites revealed no statistically significant difference in the level of
B. anthracis
spore contamination. Therefore, the immediate area around a suspected anthrax carcass should be considered substantially contaminated regardless of the condition of the carcass.
Chemotaxonomic Identification of Single Bacteria by Micro-Raman Spectroscopy: Application to Clean-Room-Relevant Biological Contaminations
Authors: Rösch Petra , Harz Michaela , Schmitt Michael , Peschke Klaus-Dieter , Ronneberger Olaf , Burkhardt Hans , Motzkus Hans-Walter , Lankers Markus , Hofer Stefan , Thiele Hans , Popp Jürgen
Published: March 2005

Microorganisms, such as bacteria, which might be present as contamination inside an industrial food or pharmaceutical clean room process need to be identified on short time scales in order to minimize possible health hazards as well as production downtimes causing financial deficits. Here we describe the first results of single-particle micro-Raman measurements in combination with a classification method, the so-called support vector machine technique, allowing for a fast, reliable, and nondestructive online identification method for single bacteria.
Environmental DNA Fragment Conferring Early and Increased Sporulation and Antibiotic Production in
Streptomyces
Species
Authors: Martinez Asuncion , Kolvek Steven J. , Hopke Joern , Tiong Yip Choi Lai , Osburne Marcia S.
Published: March 2005

Here we describe the
rep
gene, isolated from an environmental DNA library, which when transformed into
Streptomyces
species resulted in increased production of secondary metabolites and accelerated sporulation. We show that
Streptomyces lividans
strains bearing
rep
are particularly useful as expression hosts for heterologous antibiotic production.
Heterologous Expression, Purification, and Characterization of a Highly Active Xylose Reductase from
Neurospora crassa
Authors: Woodyer Ryan , Simurdiak Michael , van der Donk Wilfred A. , Zhao Huimin
Published: March 2005

A xylose reductase (XR) gene was identified from the
Neurospora crassa
whole-genome sequence, expressed heterologously in
Escherichia coli
, and purified as a His
6
-tagged fusion in high yield. This enzyme is one of the most active XRs thus far characterized and may be used for the in vitro production of xylitol.
Comparison of Diversities ofEscherichia coliO157 Shed from a Cohort of Spring-Born Beef Calves at Pasture and in Housing
Authors: Vali Leila , Pearce Michael C. , Wisely Karen A. , Hamouda Ahmed , Knight Hazel I. , Smith Alastair W. , Amyes Sebastian G. B.
Published: March 2005

A cohort of spring-born beef calves demonstrated limited genetic and phenotypic diversity ofEscherichia coliO157 when kept in a state of isolation. Despite this, there was a difference in the pulsed-field gel electrophoresis and phage types of isolates shed by cattle at pasture compared with those shed by the same cattle when weaned and housed.
Effect of Batch-Process Solar Disinfection on Survival of
Cryptosporidium parvum
Oocysts in Drinking Water
Authors: Méndez-Hermida F. , Castro-Hermida J. A. , Ares-Mazás E. , Kehoe S. C. , McGuigan K. G.
Published: March 2005

The results of batch-process solar disinfection (SODIS) of
Cryptosporidium parvum
oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m
−2
) at 40°C. Viability assays (4′,6′-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.
Genetic Diversity in the Lactose Operons of
Lactobacillus helveticus
Strains and Its Relationship to the Role of These Strains as Commercial Starter Cultures
Authors: Callanan M. J. , Beresford T. P. , Ross R. P.
Published: March 2005

Two novel insertion sequence elements, ISLhe
1
and ISLhe
15
, were located upstream of the genes encoding the β-galactosidase enzyme in
Lactobacillus helveticus
commercial starter strains. Strains with the IS
982
family element, ISLhe
1
, demonstrated reduced β-galactosidase activity compared to the
L. helveticus
type strain, whereas strains with the ISLhe
15
element expressed β-galactosidase in the absence of lactose.
Probiotic Bacteria May Become Dormant during Storage
Authors: Lahtinen Sampo J. , Gueimonde Miguel , Ouwehand Arthur C. , Reinikainen Johanna P. , Salminen Seppo J.
Published: March 2005

The determination of bacterial viability in probiotic products is of economic, technological, and clinical significance. We compared four methods to enumerate three
Bifidobacterium
strains in fermented oat products during storage. A subpopulation of nonculturable cells retained a functional cell membrane typical of viable cells, indicating that probiotic bacteria become dormant during storage.
Application of a Newly Developed ARB Software-Integrated Tool for In Silico Terminal Restriction Fragment Length Polymorphism Analysis Reveals the Dominance of a Novel
pmoA
Cluster in a Forest Soil
Authors: Ricke Peter , Kolb Steffen , Braker Gesche
Published: March 2005

TRF-CUT, an ARB-implemented tool, was developed to predict in silico the terminal restriction fragments of aligned small-subunit rRNA gene or functional gene sequences. Application of this new tool to perform directed terminal restriction fragment length polymorphism analysis of
pmoA
products obtained from a forest soil revealed that novel cluster I methanotrophic bacteria were dominant.
The Ras-MAPK signal transduction pathway, cancer and chromatin remodeling
Authors: Dunn Katherine L , Espino Paula S , Drobic Bojan , He Shihua , Davie James R
Published: March 2005

Stimulation of the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway results in a multitude of events including expression of the immediate-early genes, c-fos and c-myc. Downstream targets of this stimulated pathway are the mitogen- and stress-activated protein kinases (MSK) 1 and 2, which are histone H3 kinases. In chromatin immunoprecipitation assays, it has been shown that the mitogen-induced phosphorylated H3 is associated with the immediate-early genes and that MSK1/2 activity and H3 phosphorylation have roles in chromatin remodeling and transcription of these genes. In oncogene-transformed fibroblasts in which the Ras-MAPK pathway is constitutively active, histone H1 and H3 phosphorylation is increased and the chromatin of these cells has a more relaxed structure than the parental cells. In this review we explore the deregulation of the Ras-MAPK pathway in cancer, with an emphasis on breast cancer. We discuss the features of MSK1 and 2 and the impact of a constitutively activated Ras-MAPK pathway on chromatin remodeling and gene expression.Key words: Ras, mitogen-activated protein kinase signal transduction pathway, histone H3 phosphorylation, MSK1, breast cancer.
Analysis of an approach to oviduct-specific expression of modified chicken lysozyme genes
Authors: Shimizu Mamiko , Losos Jan K , Gibbins Ann M. Verrinder
Published: March 2005

The –2.7 kb enhancer (E) element of the chicken lysozyme gene domain appears to govern expression of the gene in macrophages but not in oviduct tubular gland cells, the only other site of lysozyme expression. The ultimate goal of our research was to determine whether lysozyme domain variants could be developed that would mainly be expressed in the oviduct so that transgenic birds could be produced that would deposit exogenous protein in the egg white. Accordingly, precise mutations were made by poxvirus-mediated gene targeting in FEF/PU.1 and CCAAT/enhancer-binding protein (C/EBP) transcription factor binding sites in the –2.7 kb E of cloned copies of a specific lysozyme gene variant that includes a hydrophobic pentapeptide tail encoding sequence inserted immediately prior to the stop codon. This variant contains the entire lysozyme domain and is cloned in a λ bacteriophage vector (λDIILys-HT); the novel tail sequence enables distinction in cell-based expression systems between transcripts of the variant and those of the endogenous gene. These various lysozyme domain mutants, in bacteriophage vector form, were tested for expression in cultured chicken blastodermal cells cotransfected with plasmids encoding the transcription factors C/EBP and v-Myb. In the absence of these plasmids, barely detectable levels of endogenous lysozyme gene transcription resulted in the blasto dermal cells. In the presence of the plasmids, however, transcripts of the endogenous gene could be detected as well as varying levels (as evaluated by quantitative real-time PCR) of transcripts of all of the lysozyme domain mutants. These results are discussed in the context of the known role and occurrence of various transcription factors involved in gene expression in differentiating macrophage cells. The ultimate test of expression of the variants in macrophages vs. oviduct cells will be to use them to produce transgenic birds.Key words: lysozyme, macrophage-specific gene expression, poxvirus-mediated gene targeting.
Single-Molecule RNA Science
Authors: Zhuang Xiaowei
Published: March 2005

▪ Abstract  The development of single-molecule detection and manipulation has allowed us to monitor the behavior of individual biological molecules and molecular complexes in real time. This approach significantly expands our capability to characterize complex dynamics of biological processes, allowing transient intermediate states and parallel kinetic pathways to be directly observed. Exploring this capability to elucidate complex dynamics, recent single-molecule experiments on RNA folding and catalysis have improved our understanding of the folding energy landscape of RNA and allowed us to better dissect complex RNA catalytic reactions, including translation by the ribosome.
Myosin lever disposition during length oscillations when power stroke tilting is reduced
Authors: Griffiths P. J. , Bagni M. A. , Colombini B. , Amenitsch H. , Bernstorff S. , Ashley C. C. , Cecchi G.
Published: March 2005

M3 reflection intensity (IM3) from tetanized, intact skeletal muscle fiber bundles was measured during sinusoidal length oscillations at 2.8 kHz, a frequency at which the myosin motor’s power stroke is greatly reduced. IM3 signals were approximately sinusoidal, but showed a “double peak” distortion previously observed only at lower oscillation frequencies. A tilting lever arm model simulated this distortion, where IM3 was calculated from the molecular structure of myosin subfragment 1 (S1). Simulations showed an isometric lever arm disposition close to normal to the filament axis at isometric tension, similar to that found using lower oscillation frequencies, where the power stroke contributes more toward total S1 movement. Inclusion of a second detached S1 in each actin-bound myosin dimer increased simulated IM3 signal amplitude and improved agreement with the experimental data. The best agreement was obtained when detached heads have a fixed orientation, insensitive to length changes, and similar to that of attached heads at tetanus plateau. This configuration also accounts for the variations in relative intensity of the two main peaks of the M3 reflection substructure after a length change. This evidence of an IM3 signal distortion when power stroke tilting is suppressed, provided that a large enough amplitude of length oscillation is used, is consistent with the tilting lever arm model of the power stroke.
Lower force and impaired performance during high-intensity electrical stimulation in skeletal muscle of GAMT-deficient knockout mice
Authors: Kan H. E. , Buse-Pot T. E. , Peco R. , Isbrandt D. , Heerschap A. , de Haan A.
Published: March 2005

Force characteristics of skeletal muscle of knockout mice lacking creatine (Cr) due to a deletion of guanidinoacetate methyltransferase (GAMT) were studied in situ. Medial gastrocnemius muscles of anesthetized GAMT-deficient (GAMT−/−) and control (Con) littermates were stimulated at optimum length via the sciatic nerve at different stimulation frequencies (60–250 Hz). GAMT−/− mice showed reduced maximal tetanic and twitch force, reduced relative force at 60 Hz, and increased relaxation times. High-intensity fatigue protocols consisting of 30 successive isometric or dynamic contractions showed a strong reduction in force at the beginning of the series in GAMT−/− mice, followed by a smaller reduction compared with Con littermates toward the end of the series. Cr supplementation for 2 days in GAMT−/− animals (GAMT[Formula: see text]) resulted in normalization to Con values for relaxation times, relative force at lower stimulation frequencies, and relative force during 30 isometric contractions. Force per muscle mass, however, remained decreased. Furthermore,GAMT[Formula: see text] mice showed differences compared with both Con and unsupplemented animals in maximal rates of force rise and relaxation times during the isometric protocol as well as in force during the dynamic protocol. Our results show that the absence of Cr plays a direct role in relaxation times, maximal rate of force rise, and force production during high-intensity fatigue protocols. The lower force per muscle mass, however, is probably caused by other factors; i.e., high intracellular guanidinoacetate concentrations.
Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit
Authors: Liu Chunming , Varnum Michael D.
Published: March 2005

Progressive cone dystrophies are a genetically heterogeneous group of disorders characterized by early deterioration of visual acuity and color vision, together with psychophysical and electrophysiological evidence of abnormal cone function and cone degeneration. Recently, three mutations in the gene encoding the CNGA3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels have been linked to progressive cone dystrophy in humans. To investigate the functional consequences of these mutations, we expressed mutant human CNGA3 subunits in Xenopus oocytes, alone or together with human CNGB3, and studied these channels using patch-clamp recording. Compared with wild-type channels, homomeric and heteromeric channels containing CNGA3-N471S or CNGA3-R563H subunits exhibited an increase in apparent affinity for cGMP and an increase in the relative agonist efficacy of cAMP compared with cGMP. In contrast, R277C subunits did not form functional homomeric or heteromeric channels. Cell surface expression levels, determined using confocal microscopy of green fluorescent protein-tagged subunits and patch-clamp recording, were significantly reduced for both R563H and R277C but unchanged for N471S. Overall, these results suggest that the plasma membrane localization and gating properties of cone CNG channels are altered by progressive cone dystrophy-associated mutations, providing evidence that supports the pathogenicity of these mutations.
Application of Nucleus Pulposus to L5Dorsal Root Ganglion in Rats Enhances Nociceptive Dorsal Horn Neuronal Windup
Authors: Cuellar J. M. , Montesano P. X. , Antognini J. F. , Carstens E.
Published: March 2005

Herniation of the nucleus pulposus (NP) from lumbar intervertebral discs commonly results in radiculopathic pain possibly through a neuroinflammatory response. NP sensitizes dorsal horn neuronal responses, but it is unknown whether this reflects a central or peripheral sensitization. To study central sensitization, we tested if NP enhances windup—the progressive increase in the response of a nociceptive spinal neuron to repeated electrical C-fiber stimulation—a phenomenon that may partly account for temporal summation of pain. Single-unit recordings were made from wide dynamic range (WDR; n = 36) or nociceptive-specific (NS; n = 8) L5dorsal horn neurons in 44 isoflurane-anesthetized rats. Subcutaneous electrodes delivered electrical stimuli (20 pulses, 3 times the C-fiber threshold, 0.5 ms) to the receptive field on the hindpaw. Autologous NP was harvested from a tail disc and placed onto the L5dorsal root ganglion after recording of baseline responses ( n = 22). Controls had saline applied similarly ( n = 22). Electrical stimulus trains (0.1, 0.3, and 1 Hz; 5-min interstimulus interval) were repeated every 30 min for 3–6 h after each treatment. The total number of evoked spikes (summed across all 20 stimuli) to 0.1 Hz was enhanced 3 h after NP, mainly in the after-discharge (AD) period (latency > 400 ms). Total responses to 0.3 and 1.0 Hz were also enhanced at ≥60 min after NP in both the C-fiber (100- to 400-ms latency) and AD periods, whereas the absolute windup (C-fiber + AD − 20 times the initial response) increased at ≥90 min after treatment. In saline controls, windup was not enhanced at any time after treatment for any stimulus frequency, although there was a trend toward enhancement at 0.3 Hz. These results are consistent with NP-induced central sensitization. Mechanical responses were not significantly enhanced after saline or NP treatment. We speculate that inflammatory agents released from (or recruited by) NP affect the dorsal root ganglion (and/or are transported to cord) to enhance primary afferent excitation of nociceptive dorsal horn neurons.
Glycine Binding Site of the Synaptic NMDA Receptor in Subpostremal NTS Neurons
Authors: Baptista Vander , Varanda Wamberto Antonio
Published: March 2005

The nucleus of the tractus solitarius (NTS) plays an important role in the control of several autonomic reflex functions and has glutamate and GABA as main neurotransmitters. In this work, we used patch-clamp recordings in transverse slice preparations from rats to study whether the glycine binding site of the N-methyl-d-aspartate (NMDA) receptor is saturated or not in neurons of the subpostremal NTS. Except at hyperpolarized voltages and close to the reversal potential, glycine potentiated the NMDA responses in a concentration-dependent manner. The total charge transferred by glutamatergic currents was enhanced by glycine (500 μM; from 28 ± 13 to 42 ± 18 pC at +50 mV, n = 7, P < 0.05). Glycine increased the conductance of the postsynaptic membrane, without altering its reversal potential, both in the presence (from 2.4 ± 0.06 to 3.4 ± 0.09 nS; n = 7) and absence (from 3.1 ± 0.06 to 4.4 ± 0.10 nS; n = 8) of Mg2+ in the bathing solution. d-serine, in the presence of strychnine, also increased the amplitude of the NMDA component (by 68 ± 19%, P < 0.05, n = 5). The membrane potential was hyperpolarized (16 ± 6 mV, n = 8) by glycine, suggesting the presence of inhibitory glycinergic receptors. Our results indicate that the glycine site of the NMDA receptor in neurons of the subpostremal NTS is not saturated and that glycine may act as a modulator of the NMDA transmission in this nucleus.
New Developments in the Field of Reaction Technology: The Multiparallel Reactor HPMR 50‐96
Authors: Allwardt Arne , Stoll Norbert , Wendler Christian , Thurow Kerstin
Published: March 2005

Catalytic high‐pressure reactions play an important role in classic bulk chemistry. The optimization of common reactions, the search for new and more effective catalysts, and the increasing use of catalytic pressure reactions in the field of drug development call for high‐parallel reaction systems. A crucial task of current developments, apart from the parameters of pressure, temperature, and number of reaction chambers, is, in this respect, the systems′ integration into complex laboratory
automation environments.
Diversity of a major repetitive DNA sequence in diploid and polyploid Triticeae
Authors: Contento A. , Heslop-Harrison J.S. , Schwarzacher T.
Published: March 2005

About 90 members of a major tandemly repeated DNA sequence family originally described in rye as pSc119.2 have been isolated from 11 diploid and polyploid Triticeae species using primers from along the length of the sequence for PCR amplification. Alignment and similarity analysis showed that the 120-bp repeat unit family is diverse with single nucleotide changes and few insertions and deletions occurring throughout the sequence, with no characteristic genome or species-specific variants having developed during evolution of the extant genomes. Fluorescent in situ hybridization showed that each of the large blocks of the repeat at chromosomal sites harboured many variants of the 120-bp repeat. There were substantial copy number differences between genomes, with abundant sub-terminal sites in rye, interstitial sites in the B genome of wheat, and relatively few sites in the A and D genome. We conclude that sequence homogenization events have not been operative in this repeat and that the common ancestor of the Triticeae tribe had multiple sequences of the 120-bp repeat with a range of variation not unlike that seen within and between species today. This diversity has been maintained when sites are moved within the genome and in all species since their divergence within the Triticeae.
3D Analysis of chromosome architecture: advantages and limitations with SEM
Authors: Wanner G. , Schroeder-Reiter E. , Formanek H.
Published: March 2005

Three-dimensional mitotic plant chromosome architecture can be investigated with the highest resolution with scanning electron microscopy compared to other microscopic techniques at present. Specific chromatin staining techniques making use of simultaneous detection of back-scattered electrons and secondary electrons have provided conclusive information on the distribution of DNA and protein in barley chromosomes through mitosis. Applied to investigate the structural effects of different preparative procedures, these techniques were the groundwork for the “dynamic matrix model” for chromosome condensation, which postulates an energy-dependent process of looping and bunching of chromatin coupled with attachment to a dynamic matrix of associated protein fibers. Data from SEM analysis shows basic higher order chromatin structures: chromomeres and matrix fibers. Visualization of nanogold-labeled phosphorylated histone H3 (ser10) with high resolution on chromomeres shows that functional modifications of chromatin can be located on structural elements in a 3D context.
High-resolution physical mapping of the secalin-1 locus of rye on extended DNA fibers
Authors: Yamamoto M. , Mukai Y.
Published: March 2005

High-resolution mapping of secalin-1 (Sec-1) locus has been performed by fluorescence in situ hybridization to extended DNA fibers of rye (Secale cereale, 2n = 14), employing DNA probes of λ phage clones containing the ω-secalin gene. The fluorescent signals to rye extended DNA fibers revealed continuous strings of 45 µm, corresponding to the size of 147 kb DNA. To determine the copy number of Sec-1 locus on DNA fibers, a 1.2-kb fragment including the entire coding region of the ω-secalin gene and a 1.0-kb fragment of the promoter region were amplified by PCR as probes for another fiber FISH. The physical position of these sequences was visualized as alternating fluorescent spots by multicolor in situ hybridization. Alternating signals of two DNA probes reflected the tandem repeated organization of the Sec-1 locus having 15 copies of the gene. The present findings based on fiber FISH analysis support the contention that the ω-secalin genes are arranged in a head-to-tail fashion separated by 8 kb of spacer sequences with a total length of 145 kb.
Ribosomal DNA heterochromatin in plants
Authors: Neves N. , Delgado M. , Silva M. , Caperta A. , Morais-Cecílio L. , Viegas W.
Published: March 2005

The aim of this review is to integrate earlier results and recent findings to present the current state-of-the-art vision concerning the dynamic behavior of the ribosomal DNA (rDNA) fraction in plants. The global organization and behavioral features of rDNA make it a most useful system to analyse the relationship between chromatin topology and gene expression patterns. Correlations between several heterochromatin fractions and rDNA arrays demonstrate the heterochromatic nature of the rDNA and reveal the importance of the genomic environment and of developmental controls in modulating its dynamics.
The positioning of rye homologous chromosomes added to wheat through the cell cycle in somatic cells untreated and treated with colchicine
Authors: Corredor E. , Díez M. , Shepherd K. , Naranjo T.
Published: March 2005

The arrangement of chromosome pairs 5RL and 7R added to the wild type and the ph1b mutant line of hexaploid wheat are analyzed in 2N somatic root tip cells during the cell cycle relative to the arrangement that chromosomes 5RL show in 4N tapetal cells produced after colchicine treatment. Both homologous chromosome pairs are identified at interphase and mitosis by fluorescence in situ hybridization. In nuclei at interphase, chromosomes appear as discrete domains that show the Rabl orientation. Homologous chromosomes are predominantly non-associated and their positioning seems not to be influenced by the Ph1 gene that suppresses homoeologous meiotic pairing. This pattern of arrangement contrasts with the high level of somatic pairing that sister chromosomes show in the interphase that follows chromosome duplication induced by colchicine. Disruption of pairing observed in some 4N nuclei is produced at c-anaphase which suggests no topological redistribution of homologues during conformation of the new nucleus. Homologous chromosomes show no predominant arrangement in ellipsoidal metaphase plates, which contrasts with the preferential opposite location of homologues in human prometaphase rosettes. Differences between chromosomes in the variation of the length through the cell cycle and in the chromatin morphology when the Ph1 is absent suggest different patterns of chromatin condensation in both chromosomes.
Understanding the cytological diploidization mechanism of polyploid wild wheats
Authors: Cuñado N. , Blazquez S. , Melchor L. , Pradillo M. , Santos J.L.
Published: March 2005

The allohexaploid Aegilops species (2n = 6x = 42), Ae. neglecta 6x (UUXtXtNN), Ae. juvenalis (DcDcXcXcUU), and Ae. vavilovii (DcDcXcXcSsSs) regularly form bivalents at metaphase I. However, in Ae. crassa 6x (DcDcXcXcDD) 0.27 quadrivalents per cell were observed probably as a consequence of the partial homology displayed by the D and Dc genomes. Likewise, the synthetic amphiploid Ae. ventricosa-Secale cereale (DDNNRR) is fertile and displays a diploid-like behavior at metaphase I, despite its recent origin. The pattern of synapsis at late zygotene and pachytene in the natural and artificial allohexaploids was analyzed by whole-mount surface-spreading of synaptonemal complexes under an electron microscope. It revealed that chromosomes were mostly associated as bivalents in all cases, the mean of multivalents per nucleus ranging from 0.17 (Ae. neglecta 6x) to 1.03 (Ae. crassa 6x) in the natural species and 1.05 in the Ae. ventricosa-S. cereale amphiploid. It can be concluded that the mechanism controlling bivalent formation in these species and also in the synthetic amphiploid acts mainly at zygotene by restricting synapsis to homologous chromosomes, but also acts at pachytene by preventing chiasma formation in the homoeologous associations. These observations are discussed in relation to the origin and evolution of the mechanism of diploidization in the allopolyploid species of the Poaceae family.
Synaptic behaviour of hexaploid wheat haploids with different effectiveness of the diploidizing mechanism
Authors: Martinez M. , Cuadrado C. , Laurie D.A. , Romero C.
Published: March 2005

Haploids of three cultivars of Triticum aestivum (Thatcher, Chris, and Chinese Spring) were obtained from crosses with Zea mays. The level of chromosome pairing at metaphase I and the synaptic behaviour at prophase I was studied. There were differences in the meiotic behaviour of the haploids from different cultivars. Thatcher and Chris haploids had significantly higher levels of pairing at metaphase I than Chinese Spring haploids. This metaphase I pairing was correlated with higher levels of synapsis achieved in the Thatcher and Chris prophase I nuclei than in the Chinese Spring nuclei. Variation in the effectiveness of the diploidizing mechanism among cultivars of wheat is proposed to have a genetic origin and the role of the Ph1 locus in the different haploids is discussed.
Meiotic mutations in rye Secale cereale L.
Authors: Sosnikhina S.P. , Mikhailova E.I. , Tikholiz O.A. , Priyatkina S.N. , Smirnov V.G. , Dadashev S.Y. , Kolomiets O.L. , Bogdanov Y.F.
Published: March 2005

Spontaneous meiotic mutations of winter rye Secale cereale L. (2n = 14) were revealed in inbred F2 progenies, which were obtained by self-pollination of F1 hybrids resulting from crosses of individual plants of cultivar Vyatka or weedy rye with plants of self-fertile inbred lines. The mutations cause partial or complete sterility, and are maintained in heterozygote condition. Six types of mutations were distinguished as the result of cytological analysis of meiosis and genetic analysis. (1) Plants with nonallelic asynaptic mutations sy1 and sy9 lacked bivalents in 96.8 and 67.0% metaphase I cells, respectively, formed only axial elements but not the mature synaptonemal complex (SC), and had defects in telomere clustering in early prophase I. (2) Weak asynaptic mutant sy3 showed incomplete synapsis at the start of SC degradation at diplotene and lower chiasma number; yet only 2% meiocytes lacked bivalents in MI. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19 caused nonhomologous synapsis; i.e., a varying number of univalents and occasional multivalents were observed in MI, which was preceded by switches of pairing partners and fold-back synapsis at mid-prophase I. (4) Mutation mei6 led to the formation of protrusions and minor branched structures of the SC lateral elements. (5) Allelic mutations mei8 and mei8-10 caused irregular chromatin condensation along the chromosome length in prophase I, which was accompanied by chromosome sticking and fragmentation in MI. (6) Allelic mutations mei5 and mei10 determined chromosome supercondensation, caused the disturbance of meiotic spindle assembly, arrested meiosis at various stages but did not affect formation of the pollen wall, thus arrested meiocytes got covered with the pollen wall. Analysis of double mutants revealed recessive epistatic interactions for some mutations; the epistatic group was sy9 > sy1 > sy3 > sy19. This reflects the sequence of meiotic events controlled by the corresponding genes. The expression of sy2 and sy19 proved to be modified by additional genes. Most meiotic mutations found in rye have analogs in other plants.