bluuebunny/crossref_metadata_2025 huggingface dataset:
442 ULTRASONOGRAPHIC TESTICULAR ECHOTEXTURE IN NELORE BULLS
Authors: Costa D. S. , Faria F. J. C. , Borges J. C.
Published: August 2010

The objective of this research was to determine the minimum area representative of the testicular parenchyma echotexture in Nelore bulls, and to evaluate the presence of echotexture homogeneity between the right and left testicles and between different regions of testicle. Twenty-nine Nelore bulls raised under extensive management and free of reproductive disorders were used. The average body weight was 352.8 ± 51.0 kg and age ranged between 18 and 24 months. Echotexture was assessed using a portable ultrasonography device equipped with a 7.5-MHz linear transducer. All measurement was done by a single, experienced operator, and scanner settings that affect image attributes (i.e. proximal, distal, and total gains; focus area; brightness and contrast) were standardized to predetermined values. In the examinations, a layer of gel was applied to the transducer scanning area, which was positioned in the testicular longitudinal plane, proceeding with enough pressure to capture an image. The frozen images were obtained with minimal artifacts (refraction, dispersion, and attenuation of ultrasound waves) taking care to avoid the mediastinum testis and tunica albuginea, and recorded as .bmp files in a USB pen drive connected to the ultrasound device. Echotexture was defined in terms of mean pixel value quantified using values from 0 (black) to 255 (white) through the ImageJ 1.41 software. After the selection of a specific area of testicular parenchyma, the average tonality of the gray pixels in the selected region was calculated by the software and expressed in 256 gray-scales. Squares selections of 400 (20 × 20), 1600 (40 × 40), 3600 (60 × 60), and 6400 (80 × 80) pixels were assessed from images of the extremitas capitata, middle, and extremitas caudata regions of both testicles to determine the minimum sampling area of an image needed to represent the echotexture of testicular parenchyma. The normal distribution verification (Lilliefors test) of the variable mean pixel value was tested in each area, followed by variance analysis. The averages were compared through Tukey’s test with 5% significance. There was no significant difference between the different pixel areas assessed (P > 0.05), indicating that all were samples representative of testicular echotexture. Furthermore, the extremitas capitata, middle, and extremitas caudata regions showed no significant difference in echotexture (P > 0.05) regardless of the area analyzed, and there was no significant difference between the right and left testicles (P > 0.05) in any of the assessed areas. In conclusion, this research shows that testicular echotexture assessments can be made in Nelore bulls through selections of 400, 1600, 3600, or 6400 pixels, and that images can be captured from any region of any testicle.

Fundect - Fundação deApoio ao Desenvolvimento do Ensino, CiÊncia e Tecnologia do Estado de Mato Grosso do Sul.
59 PRODUCTION OF HANDMADE CLONED GOAT EMBRYOS WITH TWO TYPES OF DONOR CELLS AND CULTURED IN THREE MEDIA
Authors: Jena M. K. , Malakar D. , De A. K. , Garg S. , Akshey Y. S.
Published: August 2010

The study was carried out to see the developmental efficiency of handmade cloned goat embryos with 3 different media: RVCL (Research Vitro Cleave, Cook, Brisbane, Australia), EDM (Embryo Development Media) and modified SOF (mSOF) and 2 types of donor cells: fetal fibroblast and adult fibroblast. Oocytes were isolated from abattoir goat ovaries, matured in maturation medium, and incubated in 5% CO2 in air at 38.5°C for 24 h. Then, the oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona free by pronase (2 mg mL-1). Protrusion cone formation in oocytes was found 95 to 100% in T20 (TCM-199 + 20% FBS). The zona-free oocytes were bisected with an ultra-sharp micro blade on the basis of visible protrusion cones on the surface of oocytes using T20 medium containing 2.5 μg mL-1 cytochalasin-B. Fetal and adult fibroblast cells were used from confluent monolayer at passage 5 after trypsinizing in 0.25% trypsin-EDTA. One somatic cell was attached with one enucleated demioocyte by phytohemagglutinin and further fused with another enucleated demioocyte through electric pulse with a combination of alternating current (4 V) and direct current (2.10 kV cm-1 for 5 μs with a single pulse) in fusion medium (0.3 M mannitol, 0.1 mM MgCl2, 0.05 mM CaCl2, and 3 mg mL-1 BSA). Then, triplets were chemically activated with 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h and cultured in the 3 media. Cleavage and morulae formation were observed at Day 7 from 183 triplets with fetal fibroblasts as donor cells in media RVCL (78.60 ± 2.23, 38.97 ± 2.1), mSOF (72.62 ± 1.89, 33.81 ± 1.9), and EDM (73.96 ± 1.66, 26.20 ± 2.04), respectively. Simultaneously, cleavage and morulae formation were observed at Day 7 from 203 triplets with adult fibroblasts as donor cells in media RVCL (73.97 ± 3.57, 33.14 ± 2.68), mSOF (76.22 ± 4.36, 26.15 ± 0.99), and EDM (65.97 ± 3.11, 20.78 ± 2.77), respectively. Among the 3 media, morulae formation was significantly higher in RVCL. Hence, in the subsequent experiment, RVCL medium was used exclusively in culture for 172 triplets. Cleavage and morulae formation at Day 7 was not significantly different (P < 0.05) in 2 types of donor cells; fetal fibroblasts (77.46 ± 3.65, 38.70 ± 2.66) and adult fibroblasts (75.74 ± 3.04, 33.77 ± 1.43), respectively. The data were analyzed using SYSTAT 7.0 (Systat, SPSS Inc., Chicago, IL, USA) after arcsine transformation, one-way ANOVA followed by Fisher’s LSD test. PCR analysis was performed with highly polymorphic 286-bp fragment of MHC-II DRB gene of cloned embryo and its donor cell. Similar bands were observed in both the cloned embryos and fibroblast cells in agar gel electrophoresis. In conclusion, development of handmade cloned embryos was higher in RVCL medium compared with the other two media tested, and efficiency of morulae formation was similar in both types of donor cells. Further study is required to optimize blastocyst production.
74 PROTEOMIC PROFILING DURING FETAL DEVELOPMENT OF BOVINE CLONES
Authors: Picard B. , Meunier B. , Heyman Y. , Chavatte-Palmer P. , Cassar-Malek I.
Published: August 2010

Previous data have shown that bovine clones display a delay in their muscle differentiation during their first year postnatal (Jurie et al. 2009 Animal 2, 244-250). This delay could originate from perturbations in fetal muscle development as illustrated by lower numbers and degree of organization of the first generation of myotubes at 60 dpc and by their lower energy metabolism and their myosin heavy chain pattern at 260 dpc (Cassar-Malek et al. 2009 Proc. XIth ISRP abst). In order to understand the mechanisms underlying the delay in myogenesis, we have performed a comparative proteomic analysis of the semitendinosus muscle in fetuses derived from somatic nuclear transfer and their control counterparts obtained after AI at these two important developmental stages. Two-dimensional electrophoresis using a 3-10 non-linear pH gradient were performed on samples at 60 dpc (in a group of Holstein animals and a group of Charolais animals, n = 4 fetuses per lot) and at 260 dpc (in a group of Holstein animals, n = 4 fetuses per lot). Gel analysis was conducted using the image analysis SameSpots (Nonlinear Dynamics, Newcastle upon Tyne, UK). As expected, the protein profiles were visually very different between developmental stages. At 60 dpc, 463 spots common to all gels were retained for statistical analysis using the significance analysis of microarrays (SAM) method (FDR <5%; Meunier et al. 2005 Anal. Biochem. 340, 226-230). At 260 dpc, 491 spots were selected for SAM analysis. The statistical analysis revealed a small number of differential spots (9 and 10 spots, respectively, at 60 dpc in Holstein and Charolais, and 10 spots at 260 dpc Holstein). The differential spots were excised from the gels and their identification by mass spectrometry is in progress. Preliminary results are presented in Table 1. In conclusion, subtle changes in the muscle proteome were detected in clones v. controls. Some of them were related to the regulation of cell cycle/apoptosis at 60 dpc and to energy metabolism and chaperone activity at 260 dpc. The relevance of these changes will be further explored using bioinformatics tools.

Table 1.Examples of identified spots with differential abundance between clones and controls

The authors thank C. Barboiron for excellent technical assistance.
84 COMPUTER-ASSISTED SPERM ANALYSIS OF FROZEN SPERM MOTILITY AFTER REPEATED EXPOSURES TO ROOM TEMPERATURE
Authors: Alvaro Garcia Guerra A. , Brogliatti G. M.
Published: August 2010

The key factorin long-term cryopreservation is the very low temperature of liquid nitrogen. Several studies suggest temperatures should be maintained at -130°C or less to avoid cell damage. Damage due to initial exposure may not be overt; however, after repeated exposures a reduction in postthaw viability may become evident (Barth A 1991 Proc. 10th Annu. Conv. Am. ET Assoc, 20-26). The CASA system provides an opportunity to assess multiple motility characteristics on a semen sample objectively and with high repeatability. An experiment was designed to evaluate the effect that repeated exposure of frozen semen in 0.5-mL straws during 15 s to room temperature produces on motility characteristics assessed by CASA system. Groups were formed according to the number of exposures per straw; groups were as follows: 0, 3, 5, and 10 times of exposure during 15 s. Thirty-two ejaculates from different bulls (15 Angus, 3 Hereford, 8 Brangus, 3 others) were diluted using a chemically semi-defined media (Andromed, Minitub, Germany) and frozen in an automatic freezer (Digicool, IMV, Paillette Crista, France). Four frozen straws per bull were used, one for each group. Straws were exposed to a room temperature (15°C ± 1.28) and then placed back into liquid nitrogen. Semen thawing was conduced in a water bath at 37°C during 1 min. Motility characteristics were evaluated by the IVOS Sperm Analyzer (Hamilton Thorne Research). Two chambers of 20 μm depth and 5 fields per chamber were analyzed (30 frames/0.5 s for each field). Seven motility parameters were evaluated: % of motile sperm; % of progressive sperm; VAP (path velocity, μms-1); VCL (track speed, μm/s); ALH (lateral amplitude, μm); BCF (beat frequency, Hz); and LIN (linearity, %). The Kruskal Wallis test was used to compare variables among groups, and results are shown in Table 1. The average temperature inside the straw after 15 s of exposure was of -122.6°C. No difference (P > 0.05) was found among the groups for any of the 7 motility parameters. In conclusion, sperm motility seems not to be affected if straws are exposed up to 10 times during 15 s to room temperature. More research should be done to test higher room temperatures and pregnancy rates after AI.

Table 1.CASA parameters of frozen sperm after different numbers of exposures at 15°C
85 THE COMPARISON OF SPERM FREEZABILITY USING TWO EGG YOLK-FREE DILUENTS IN ZANDI RAM
Authors: Ardebili R. , Towhidi A. , Zeinodini S. , Bahreini M. , Dirandeh E.
Published: August 2010

The aim of this study was to investigate the effect of 2 different commercial egg yolk-free extenders, Bioxcell and AndroMed, on post-thaw ram sperm motility and recovery rate. A total of 10 ejaculates were collected from 5 adult Iranian Zandi rams using artificial vagina during 3 weeks, once per week. Preexamination was conducted on semen, and semen motility >70% were selected. Semen samples were pooled and diluted with Bioxcell or AndroMed. Pooled semen was split into 2 treatments and extended to a final concentration of 200 × 108 spermatozoa per mL with extenders. The extended semen was manually packed into 0.25-mL mini-straws and sealed with PVC powder. After 4 h of equilibration in a waterbath at 5°C, the straws were dried with paper tissue, placed horizontally on a rack, and transferred to an isothermal box to be frozen in vapor 5 cm above liquid nitrogen. After 10 min, the straws were plunged into liquid nitrogen. Freezing was conducted using IMV semi-automatic equipment. The percentage of motility and progressive motility of sperm were evaluated before freezing (at 37°C), after refrigeration (at 5°C) and thawing, and recovery rate was calculated. To evaluate indices of sperm motility including the percentage of motile sperm and the progressive forward motility, semen was diluted with 0.9% NaCl w/v (1:100). Ten μL of diluted semen was placed on the prewarmed (37°C) microscope slide (76.2 mm × 24.5 mm; Pearl, China) covered with a cover slip (24 × 24 mm; Menzel-Glaser, Germany) and examined 10 different fields under a phase contrast microscope (Nikon, Tokyo, Japan), at ×200. Dilution factor 1:5 was used. The microscope was equipped with warm plate set at 37°C. Data were analyzed using Proc GLM of SAS. The effect of extender on sperm qualitative characteristics after thawing was significant. Least squares mean percent motility and progressive motility of AndroMed (36.05% ± 0.75; 29.0% ± 0.84) was significantly higher (P < 0.01) than Bioxcell (8.02% ± 0.35; 4.0% ± 0.60); least squares mean recovery rate in AndroMed (34.23% ± 1.05) also was higher (P < 0.01) than Bioxcell (10.0% ± 0.92). Results suggested that AndroMed in comparison with Bioxcell had more ability to preserve sperm quality during the freezing and thawing process.
98 EIGHT-CELL STAGE DELINEATES CRYOTOLERANCE OF VITRIFIED RABBIT EMBRYOS
Authors: Lin T.-A. , Chen C.-H. , Sung L.-Y. , Ju J.-C. , Chen E. , Du F. , Xu J.
Published: August 2010

A number of groups have successfully vitrified rabbit embryos at morula or blastocyst (BL) stage. For earlier stages (1- to 8-celled), however, there are very limited studies and the results are generally unsatisfactory. In this study, we examined the survival and developmental competence of rabbit embryos vitrified at different preimplantational stages. Sexually matured female New Zealand White (NZW) rabbits were superovulated with our standard protocols, followed by mating with NZW males. At 18h post-hCG treatment, viable fertilized embryos were collected and cultured in 2.5% FBS B2 medium (Laboratories CCD, Paris, France) in 5% CO2 with humidified air at 38.5°C. A total of 691 rabbit embryos at pronuclear, 2-celled, 4-celled, 8-celled, and morula/early blastocyst (BL; Day 3) stages were vitrified by the open pulled straw (OPS) method in HEPES buffered TCM-199 medium containing 20% fetal calf serum, ethylene glycol, and dimethyl sulfoxide. After stored in liquid nitrogen for at least 1 month, embryos were sequentially warmed, rehydrated, and washed before culture in B2 culture medium. Survival and developmental rates were analyzed by general linear model analysis (SPSS 11.0, SPSS Inc., Chicago, IL, USA). Embryos vitrified at 8-celled stage or beyond showed greater survival, and expanded BL and hatching rates than those at pronuclear, 2-celled and 4-celled embryos (Table 1). In particular, the expanded and hatched BL rates were significantly higher in the 8-celled group than those in 4-celled group, suggesting that the 8-celled is the threshold stage to tolerate the vitrification procedure in rabbit embryos. In addition, the rates of expanded and hatched blastocysts in morula/BL group were still significantly higher than those in the 8-celled group. Our results may provide the proper timing of cryopreserving fertilized, transgenic, and/or cloned rabbit embryos at early stages for biomedical research.

Table 1.Survival and developmental competence of rabbit embryos vitrified at different stages after thawing

This study was supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.
Specific involvement of postsynaptic GluN2B-containing NMDA receptors in the developmental elimination of corticospinal synapses
Authors: Ohno Takae , Maeda Hitoshi , Murabe Naoyuki , Kamiyama Tsutomu , Yoshioka Noboru , Mishina Masayoshi , Sakurai Masaki
Published: August 2010

The GluN2B (GluRε2/NR2B) and GluN2A (GluRε1/NR2A) NMDA receptor (NMDAR) subtypes have been differentially implicated in activity-dependent synaptic plasticity. However, little is known about the respective contributions made by these two subtypes to developmental plasticity, in part because studies of GluN2B KO [
Grin2b
−/−
(
2b
−/−
)] mice are hampered by early neonatal mortality. We previously used in vitro slice cocultures of rodent cerebral cortex (Cx) and spinal cord (SpC) to show that corticospinal (CS) synapses, once present throughout the SpC, are eliminated from the ventral side during development in an NMDAR-dependent manner. To study subtype specificity of NMDAR in this developmental plasticity, we cocultured Cx and SpC slices derived from postnatal day 0 (P0) animals with different genotypes [
2b
−/−
,
Grin2a
−/−
(
2a
−/−
), or WT mice]. The distribution of CS synapses was studied electrophysiologically and with a voltage-sensitive dye. Synapse elimination on the ventral side was blocked in WT(Cx)-
2b
−/−
(SpC) pairs but not in WT(Cx)-
2a
−/−
(SpC) or
2b
−/−
(Cx)-WT(SpC) pairs. CS axonal regression was also observed through live imaging of CS axons labeled with enhanced yellow fluorescent protein (EYFP) through exo utero electroporation. These findings suggest that postsynaptic GluN2B is selectively involved in CS synapse elimination. In addition, the elimination was not blocked in
2a
−/−
SpC slices, where Ca
2+
entry through GluN2B-mediated CS synaptic currents was reduced to the same level as in
2b
−/−
slices, suggesting that the differential effect of GluN2B and GluN2A in CS synapse elimination might not be explained based solely on greater Ca
2+
entry through GluN2B-containing channels.
Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle
Authors: Roepke Jonathan , Salim Vonny , Wu Maggie , Thamm Antje M. K. , Murata Jun , Ploss Kerstin , Boland Wilhelm , De Luca Vincenzo
Published: August 2010

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (
Catharanthus roseus
) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them.
Clustering of dispersed ribosomal DNA and its role in gene regulation and chromosome-end associations in malaria parasites
Authors: Mancio-Silva Liliana , Zhang Qingfeng , Scheidig-Benatar Christine , Scherf Artur
Published: August 2010

Dynamic changes in gene positioning contribute to differential expression of virulence-related gene families in protozoan pathogens; however, the role of nuclear architecture in gene expression in the human malaria parasitePlasmodium falciparumremains poorly understood. Here we investigated the developmentally regulated ribosomal RNA (rRNA) gene family inP. falciparum, which, unlike that in most eukaryotes, contains only a few unlinked copies of rRNA genes scattered over the subtelomeric regions of several chromosomes. We show that active and silent members of this gene family cluster in a single perinuclear nucleolus. This rDNA nuclear confinement is DNA sequence dependent, as plasmids carrying rDNA fragments are targeted to the nucleolus. Likewise, insertion of an rDNA sequence into a subtelomere from a chromosome lacking rRNA genes leads to repositioning in the nucleolus. Furthermore, we observed that rDNA spatial organization restricted interchromosomal interactions, as chromosome end-bearing rRNA genes were found to be preferentially juxtaposed, demonstrating nonrandom association of telomeres. Using Br-UTP incorporation, we observed two α-amanitin–resistant nucleolar transcription sites that disappeared when the rDNA cluster broke up in the replicative blood stages. Taken together, our results provide conceptual insights into functionally differentiated nuclear territories and their role in gene expression in malaria parasites.
Tracing the protectors path from the germ line to the genome
Authors: Coutandin Daniel , Der Ou Horng , Löhr Frank , Dötsch Volker
Published: August 2010

One of the basic principles that nature uses in evolution is to recycle successful concepts and create new functions by modifying existing units. This conservatism in evolution has resulted in an astonishingly high sequence identity of genes, even between evolutionarily distant species such as the nematodeCaenorhabditis elegansandHomo sapiens. The recycling of successful concepts in conjunction with gene duplication events has also led to the existence of highly homologous proteins within the genome of many species. Often, these homologous proteins show similar, yet distinct functions that, in combination with their individual tissue distribution, define their specific physiological role. One prominent example is the p53 protein family, which consists of p53, p63, and p73. Recent advances in understanding the specific biological functions of these members have shed some light onto the evolution of this crucial protein family, from a germ line-specific quality-control factor to a somatic tumor suppressor. Furthermore, structures of the oligomerization domains of the mammalian paralogs, p53 and p73, and invertebrate orthologs, CEP-1 and DMP53, have delineated evolutionary changes and revealed that the oligomerization domain of p53 lacks additional stabilizing structural elements present in all other p53 family members. This suggests that p53 is the most recent evolutionary member of this protein family and predicts a mechanism for p53 activation.
Fine analysis of spontaneous MAGE-C1/CT7–specific immunity in melanoma patients
Authors: Nuber Natko , Curioni-Fontecedro Alessandra , Matter Claudia , Soldini Davide , Tiercy Jean Marie , von Boehmer Lotta , Moch Holger , Dummer Reinhard , Knuth Alexander , van den Broek Maries
Published: August 2010

Cancer/testis (CT) antigens represent prime candidates for immunotherapy in cancer patients, because their expression is restricted to cancer cells and germ cells of the testis. MAGE-C1/CT7 is a CT antigen that is highly expressed in several types of cancers. Spontaneous occurrence of CT7-specific antibodies was previously detected by SEREX screen in a melanoma patient. However, naturally occurring CT7-specific T-cell responses have thus far not been detected. Peripheral blood mononuclear cells (PBMCs) from 26 metastatic melanoma patients expressing CT7 in their tumor lesions (CT7+) were analyzed for CT7-specific T-cell responses using overlapping peptides. CT7-specific CD4+T-cell responses were detected in three patients (11.5%). These CT7-specific CD4+T-cell responses were detectable in melanoma patients’ PBMCs exclusively from preexisting CD45RA−memory CD4+T-cell pool. Additional CT7-specific memory CD4+T-cell responses were detected in CT7+melanoma patients after depletion of CD4+CD25high Treg cells showing that Treg cells impact on CT7-specific CD4+T cells in melanoma patients. CT7-specific CD4+T-cell clones were generated and used to define minimal epitopes, restriction elements, and confirm the recognition of naturally processed antigen. Surprisingly, these clones were able to secrete perforin and exert cytotoxicity. This study shows that CT7 can induce specific cellular immunity in melanoma patients. Based on these findings, CT7 will be further explored as a potential vaccine for melanoma immunotherapy.
TCF4 and CDX2, major transcription factors for intestinal function, converge on the same
cis
-regulatory regions
Authors: Verzi Michael P. , Hatzis Pantelis , Sulahian Rita , Philips Juliet , Schuijers Jurian , Shin Hyunjin , Freed Ellen , Lynch John P. , Dang Duyen T. , Brown Myles , Clevers Hans , Liu X. Shirley , Shivdasani Ramesh A.
Published: August 2010

Surprisingly few pathways signal between cells, raising questions about mechanisms for tissue-specific responses. In particular, Wnt ligands signal in many mammalian tissues, including the intestinal epithelium, where constitutive signaling causes cancer. Genome-wide analysis of DNA
cis
-regulatory regions bound by the intestine-restricted transcription factor CDX2 in colonic cells uncovered highly significant overrepresentation of sequences that bind TCF4, a transcriptional effector of intestinal Wnt signaling. Chromatin immunoprecipitation confirmed TCF4 occupancy at most such sites and co-occupancy of CDX2 and TCF4 across short distances. A region spanning the single nucleotide polymorphism rs6983267, which lies within a MYC enhancer and confers colorectal cancer risk in humans, represented one of many co-occupied sites. Co-occupancy correlated with intestine-specific gene expression and CDX2 loss reduced TCF4 binding. These results implicate CDX2 in directing TCF4 binding in intestinal cells. Co-occupancy of regulatory regions by signal-effector and tissue-restricted transcription factors may represent a general mechanism for ubiquitous signaling pathways to achieve tissue-specific outcomes.
Severe anemia in theNanmutant mouse caused by sequence-selective disruption of erythroid Krüppel-like factor
Authors: Siatecka Miroslawa , Sahr Kenneth E. , Andersen Sabra G. , Mezei Mihaly , Bieker James J. , Peters Luanne L.
Published: August 2010

Studies of mouse models of anemia have long provided fundamental insights into red blood cell formation and function. Here we show that the semidominant mouse mutationNan(“neonatal anemia”) carries a single amino acid change (E339D) within the second zinc finger of the erythroid Krüppel-like factor (EKLF), a critical erythroid regulatory transcription factor. The mutation alters the DNA-binding specificity of EKLF so that it no longer binds promoters of a subset of its DNA targets. Remarkably, even when mutantNanand wild-type EKLF alleles are expressed at equivalent levels, the mutant form selectively interferes with expression of EKLF target genes whose promoter elements it no longer binds. This interference yields a distorted genetic output and selective protein deficiencies that differ from those seen in EKLF-heterozygous and EKLF-null red blood cells and presents a unique and unexpected mechanism of inherited disease.
Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in
Escherichia coli
Authors: Cohen Susan E. , Lewis Cindi A. , Mooney Rachel A. , Kohanski Michael A. , Collins James J. , Landick Robert , Walker Graham C.
Published: August 2010

We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that
Escherichia coli nusA11
(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an
N
2
-furfuryl-dG (
N
2
-f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by
E. coli
RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the nontranscribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZ-induced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in an alternative class of TCR involved in the identification and removal of a class of lesion, such as the
N
2
-f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP.
Defective feedback regulation of NF-κB underlies Sjögren
’
s syndrome in mice with mutated κB enhancers of the
IκBα
promoter
Authors: Peng Bailu , Ling Jianhua , Lee Andrew Joon , Wang Zilai , Chang Zhe , Jin Wei , Kang Ya'an , Zhang Richard , Shim David , Wang Huamin , Fleming Jason B. , Zheng Hui , Sun Shao-Cong , Chiao Paul J.
Published: August 2010

Feedback regulation of transcription factor NF-κB by its inhibitor IκBα plays an essential role in control of NF-κB activity. To understand the biological significance of IκBα-mediated feedback regulation of NF-κB, we generated mice harboring mutated κB enhancers in the promoter of the
IκBα
gene (

IκBα
M/M

) to inhibit NF-κB–regulated IκBα expression. Here, we report that these mutant mice are defective in NF-κB–induced expression of IκBα. This defective feedback regulation of NF-κB by IκBα not only altered activity of NF-κB, but also the expression of NF-κB–regulated genes. As a result,

IκBα
M/M

, the homozygous knock-in mice with mutated κB enhancers in the
IκBα
promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjögren
’
s Syndrome. These findings therefore demonstrate that the IκBα-mediated feedback regulation of NF-κB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjögren
’
s Syndrome, and suggest the potential of NF-κB signaling as a therapeutic target for Sjögren
’
s Syndrome and other autoimmune diseases.
Imaging in-plane and normal stresses near an interface crack using traction force microscopy
Authors: Xu Ye , Engl Wilfried C. , Jerison Elizabeth R. , Wallenstein Kevin J. , Hyland Callen , Wilen Larry A. , Dufresne Eric R.
Published: August 2010

Colloidal coatings, such as paint, are all around us. However, we know little about the mechanics of the film-forming process because the composition and properties of drying coatings vary dramatically in space and time. To surmount this challenge, we extend traction force microscopy to quantify the spatial distribution of all three components of the stress at the interface of two materials. We apply this approach to image stress near the tip of a propagating interface crack in a drying colloidal coating and extract the stress intensity factor.
Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from
Escherichia coli
Authors: Bogdanov Mikhail , Heacock Philip , Guan Ziqiang , Dowhan William
Published: August 2010

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of
Escherichia coli
, when reconstituted in
E. coli
phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of
E. coli
-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of
E. coli
in which PC completely replaces PE even though the structure is not completely native.
E. coli
-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.
Physical and functional interaction between the condensin MukB and the decatenase topoisomerase IV in
Escherichia coli
Authors: Hayama Ryo , Marians Kenneth J.
Published: August 2010

Proper geometric and topological organization of DNA is essential for all chromosomal processes. Two classes of proteins play major roles in organizing chromosomes: condensin complexes and type II topoisomerases. In
Escherichia coli
, MukB, a structural maintenance of chromosome-like component of the bacterial condensin, and topoisomerase IV (Topo IV), a type II topoisomerase that decatenates the newly replicated daughter chromosomes, are both essential for chromosome segregation in rapidly growing cells. However, little is known about the interplay between MukB and Topo IV. Here we demonstrate a physical and functional interaction between MukB and ParC, a subunit of Topo IV, in vitro. The site of MukB interaction was located on the C-terminal domain of ParC and a loss-of-interaction mutant, ParC R705E R729A, was isolated. This variant retained full activity as a topoisomerase when reconstituted with ParE to form Topo IV. We show that MukB stimulates the superhelical DNA relaxation activity of wild-type Topo IV, but not that of Topo IV reconstituted with ParC R705E R729A.
Missense mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-β aggregates
Authors: Singh Surinder M. , Kongari Narsimulu , Cabello-Villegas Javier , Mallela Krishna M. G.
Published: August 2010

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations—L54R, A168D, A171P, and Y231N—on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded α-helical protein in solution, as is evident from its α-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR
15
N-
1
H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal (T
1
) and transverse (T
2
) relaxation experiments. Compared to WT, three mutants—L54R, A168D, and A171P—show a decreased α-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a “molten globule” state. In contrast, Y231N has an α-helical content similar to WT and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-β structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration.
Hyperactivation of nuclear factor of activated T cells 1 (NFAT1) in T cells attenuates severity of murine autoimmune encephalomyelitis
Authors: Ghosh Srimoyee , Koralov Sergei B. , Stevanovic Irena , Sundrud Mark S. , Sasaki Yoshiteru , Rajewsky Klaus , Rao Anjana , Müller Martin R.
Published: August 2010

Nuclear factor of activated T cells (NFAT) proteins are a group of Ca
2+
-regulated transcription factors residing in the cytoplasm of resting cells. Dephosphorylation by calcineurin results in nuclear translocation of NFAT and subsequent expression of target genes; rephosphorylation by kinases, including casein kinase 1 (CK1), restores NFAT to its latent state in the cytoplasm. We engineered a hyperactivable version of NFAT1 with increased affinity for calcineurin and decreased affinity for casein kinase 1. Mice expressing hyperactivable NFAT1 in their T-cell compartment exhibited a dramatically increased frequency of both IL-17– and IL-10–producing cells after differentiation under Th17 conditions—this was associated with direct binding of NFAT1 to distal regulatory regions of
Il-17
and
Il-10
gene loci in Th17 cells. Despite higher IL-17 production in culture, the mice were significantly less prone to myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis than controls, correlating with increased production of the immunomodulatory cytokine IL-10 and enhanced accumulation of regulatory T cells within the CNS. Thus, NFAT hyperactivation paradoxically leads to decreased susceptibility to experimental autoimmune encephalomyelitis, supporting previous observations linking defects in Ca
2+
/NFAT signaling to lymphoproliferation and autoimmune disease.
Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field
Authors: Dunse K. M. , Stevens J. A. , Lay F. T. , Gaspar Y. M. , Heath R. L. , Anderson M. A.
Published: August 2010

Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes.
Nicotiana alata
proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco,
Nicotiana alata
. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests. Although consumption of NaPI dramatically reduced the growth and development of a major insect pest,
Helicoverpa punctigera
, we discovered that surviving larvae had high levels of chymotrypsin activity resistant to inhibition by NaPI. We found a potato type I inhibitor,
Solanum tuberosum
potato type I inhibitor (StPin1A), was a strong inhibitor of the NaPI-resistant chymotrypsin activity. The combined inhibitory effect of NaPI and StPin1A on
H. armigera
larval growth in the laboratory was reflected in the increased yield of cotton bolls in field trials of transgenic plants expressing both inhibitors. Better crop protection thus is achieved using combinations of inhibitors in which one class of proteinase inhibitor is used to match the genetic capacity of an insect to adapt to a second class of proteinase inhibitor.
Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners
Authors: Schulman Sol , Wang Belinda , Li Weikai , Rapoport Tom A.
Published: August 2010

Vitamin K epoxide reductase (VKOR) sustains blood coagulation by reducing vitamin K epoxide to the hydroquinone, an essential cofactor for the γ-glutamyl carboxylation of many clotting factors. The physiological redox partner of VKOR remains uncertain, but is likely a thioredoxin-like protein. Here, we demonstrate that human VKOR has the same membrane topology as the enzyme fromSynechococcussp., whose crystal structure was recently determined. Our results suggest that, during the redox reaction, Cys43 in a luminal loop of human VKOR forms a transient disulfide bond with a thioredoxin (Trx)-like protein located in the lumen of the endoplasmic reticulum (ER). We screened for redox partners of VKOR among the large number of mammalian Trx-like ER proteins by testing a panel of these candidates for their ability to form this specific disulfide bond with human VKOR. Our results show that VKOR interacts strongly with TMX, an ER membrane-anchored Trx-like protein with a unique CPAC active site. Weaker interactions were observed with TMX4, a close relative of TMX, and ERp18, the smallest Trx-like protein of the ER. We performed a similar screen with Ero1-α, an ER-luminal protein that oxidizes the Trx-like protein disulfide isomerase. We found that Ero1-α interacts with most of the tested Trx-like proteins, although only poorly with the membrane-anchored members of the family. Taken together, our results demonstrate that human VKOR employs the same electron transfer pathway as its bacterial homologs and that VKORs generally prefer membrane-bound Trx-like redox partners.
MHC class II–restricted antigen presentation by plasmacytoid dendritic cells inhibits T cell–mediated autoimmunity
Authors: Irla Magali , Küpfer Natalia , Suter Tobias , Lissilaa Rami , Benkhoucha Mahdia , Skupsky Jonathan , Lalive Patrice H. , Fontana Adriano , Reith Walter , Hugues Stéphanie
Published: August 2010

Although plasmacytoid dendritic cells (pDCs) express major histocompatibility complex class II (MHCII) molecules, and can capture, process, and present antigens (Ags), direct demonstrations that they function as professional Ag-presenting cells (APCs) in vivo during ongoing immune responses remain lacking. We demonstrate that mice exhibiting a selective abrogation of MHCII expression by pDCs develop exacerbated experimental autoimmune encephalomyelitis (EAE) as a consequence of enhanced priming of encephalitogenic CD4+ T cell responses in secondary lymphoid tissues. After EAE induction, pDCs are recruited to lymph nodes and establish MHCII-dependent myelin-Ag–specific contacts with CD4+ T cells. These interactions promote the selective expansion of myelin-Ag–specific natural regulatory T cells that dampen the autoimmune T cell response. pDCs thus function as APCs during the course of EAE and confer a natural protection against autoimmune disease development that is mediated directly by their ability to present of Ags to CD4+ T cells in vivo.
Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination
Authors: Shih Han-Yu , Krangel Michael S.
Published: August 2010

Studies have suggested that antigen receptor loci adopt contracted conformations to promote long-distance interactions between gene segments during V(D)J recombination. The Tcra/Tcrd locus is unique because it undergoes highly divergent Tcrd and Tcra recombination programs in CD4−CD8− double negative (DN) and CD4+CD8+ double positive (DP) thymocytes, respectively. Using three-dimensional fluorescence in situ hybridization, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus. We found that the 3′ portion of the locus is contracted in DN and DP thymocytes but not in B cells. Remarkably, the 5′ portion of the locus is contracted in DN thymocytes but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving Vδ gene segments distributed over 1 Mb, whereas the unique 3′-contracted, 5′-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3′ of the available Vα gene segments. This would maintain a large pool of distal 5′ Vα gene segments for subsequent rounds of recombination. Thus, distinct contracted conformations of the Tcra/Tcrd locus may facilitate a transition from a Tcrd to a Tcra mode of recombination during thymocyte development.
Mutant p53 facilitates somatic cell reprogramming and augments the malignant potential of reprogrammed cells
Authors: Sarig Rachel , Rivlin Noa , Brosh Ran , Bornstein Chamutal , Kamer Iris , Ezra Osnat , Molchadsky Alina , Goldfinger Naomi , Brenner Ori , Rotter Varda
Published: August 2010

p53 deficiency enhances the efficiency of somatic cell reprogramming to a pluripotent state. As p53 is usually mutated in human tumors and many mutated forms of p53 gain novel activities, we studied the influence of mutant p53 (mut-p53) on somatic cell reprogramming. Our data indicate a novel gain of function (GOF) property for mut-p53, which markedly enhanced the efficiency of the reprogramming process compared with p53 deficiency. Importantly, this novel activity of mut-p53 induced alterations in the characteristics of the reprogrammed cells. Although p53 knockout (KO) cells reprogrammed with only Oct4 and Sox2 maintained their pluripotent capacity in vivo, reprogrammed cells expressing mutant p53 lost this capability and gave rise to malignant tumors. This novel GOF of mut-p53 is not attributed to its effect on proliferation, as both p53 KO and mut-p53 cells displayed similar proliferation rates. In addition, we demonstrate an oncogenic activity of Klf4, as its overexpression in either p53 KO or mut-p53 cells induced aggressive tumors. Overall, our data show that reprogrammed cells with the capacity to differentiate into the three germ layers in vitro can form malignant tumors, suggesting that in genetically unstable cells, such as those in which p53 is mutated, reprogramming may result in the generation of cells with malignant tumor-forming potential.
Influence of grain refinement and induced crystal defects on the magnetic properties of Ni50Mn36Sn14 Heusler alloy
Authors: Alves A L , Passamani E C , Nascimento V P , Takeuchi A Y , Larica C
Published: August 2010

The effect of high energy ball milling on the structural, magnetic and magnetocaloric properties of Ni50Mn36Sn14 Heusler-type alloy has been studied. X-ray diffraction results have revealed a reduction in the crystalline grain size concomitantly with defect inclusions in the crystalline lattice, favouring a chemical disorder effect that transforms the L21–B2-type disordered structure to a simple cubic B2-type structure for increasing milling time. From magnetometry and Mössbauer spectroscopy results, a decrease in the ferromagnetic exchange interaction contribution, an enhancement of the effective exchange bias field and a significant reduction in the magnetic entropy change of the milled alloy are observed.