bluuebunny/crossref_metadata_2025 huggingface dataset:
Total glutamine content in human milk is not influenced by gestational age
Authors: Jochum Frank , Colling Stephanie , Meinardus Pamela , Alteheld Birgit , Stehle Peter , Fusch Christoph
Published: June 2006

Background: Glutamine may be a conditionally indispensable amino acid in neonates and should be given in adequate amounts. Reliable information concerning the glutamine content of human milk is lacking. Aim: To assess total glutamine content in human milk using a novel analytical procedure, and to evaluate the potential influence of time of delivery and lactational stage. Methods: The content of free and protein‐bound glutamine was assessed in transitional (days 4–7 of lactation) and mature (days 29–34) human milk, after preterm (<35 wk of gestation, n=20) or term (>37 wk, n=20) delivery. Milk samples were obtained by manual expression and stored at ≤70°C. Measurement of protein‐bound glutamine was performed after stabilization by bis(1,1‐trifluoroacetoxy)iodobenzene (BTI). Free glutamine was detected by HPLC. Results: There was no difference concerning glutamine content in human milk after term or preterm delivery (median of 5000 vs 4960 µmol/l milk). The protein‐bound glutamine content decreased with the duration of lactation (6230 vs 4540 µmol/l milk). Free glutamine accounted for only 3–10% of the protein‐bound glutamine content. The free glutamine content showed a high variability after preterm and term delivery.

Conclusion: This study presents for the first time data on glutamine intake in preterm and term neonates fed human milk. With these data, it is possible to formulate adequately composed nutritional therapy for infants at risk of glutamine imbalance.
Sleep duration and overweight among Australian children and adolescents
Authors: Eisenmann Joey C. , Ekkekakis Panteleimon , Holmes Megan
Published: June 2006

Aim: To examine the association between sleep and overweight and waist circumference (WC) in children and adolescents. Methods: Data were from a nationally representative sample of 6324 7–15‐y‐old males and females from the Australian Health and Fitness Survey. Associations between sleep duration and body mass index (BMI) and WC were examined by analysis of covariance, linear regression, and logistic regression. Results: In the total sample, there was a significant main effect across sleep‐duration categories (≤8 h, 8–9 h, 9–10 h, and ≥10 h) for BMI. Linear regression showed significant age, sex, age‐sleep, and age‐sex‐sleep interactions in the total sample. There was an inverse graded relationship between sleep and BMI and WC in boys. In boys, there was a 1.6–1.8 times greater odds of overweight for those who reported 8–9 h or 9–10 h of sleep compared to those reporting ≥10 h of sleep. For boys reporting ≤8 h of sleep, there was about 3.1 times greater odds of overweight compared to those reporting ≥10 h of sleep. In contrast, no significant associations between sleep and overweight were found in girls.

Conclusion: Sleep duration was inversely related with overweight in young males but not females.
Duration of breastfeeding among first‐time mothers in the United States: Results of a national survey
Authors: Taylor Julie Scott , Risica Patricia Markham , Geller Lauren , Kirtania Usree , Cabral Howard J.
Published: June 2006

Aim: To determine the characteristics of primiparous women who breastfeed for only short periods of time. Methods: The 2002 National Survey of Family Growth was used to analyze the characteristics of a national probability sample of 3229 first‐time US mothers aged 15 to 44 y with children ages 1 to 18 y old, 1960 of whom breastfed. Main outcome measures in the cross‐sectional study included breastfeeding at birth and at 3 mo as determined by in‐person, computer‐assisted interviews conducted by trained female interviewers. Results: Sixty‐two percent of first‐time mothers with singleton live births initiated any breastfeeding and 36% were still breastfeeding at 3 mo. Those who were older, married, and at higher educational levels were significantly more likely to continue breastfeeding beyond 3 mo. These same demographic factors were associated with differences in breastfeeding rates both early (within the first week of life) and consistently over the first 3 mo. In multivariate analyses, only educational level remained as a predictor of breastfeeding.

Conclusion: Targeted breastfeeding interventions for young, single, less‐educated women are warranted in a culturally sensitive context, both prenatally and immediately postpartum, to improve the duration of breastfeeding among first‐time mothers in the US.
Neonatal behaviour characteristics and psychological problems at 6 years
Authors: Canals Josepa , Esparó Griselda , Fernández‐Ballart Joan D.
Published: June 2006

Aim: To examine the predictive capacity of the Neonatal Behavioral Assessment Scale (NBAS) on psychological problems at the age of 6 y. Methods: Eighty full‐term infants of optimal health were evaluated at 3 d and 4 wk of age with the NBAS and at 6 y with the Child Behavior Checklist (CBCL) and the Inattention‐Overactivity with Aggression Conners Teacher's Rating Scale (IOWA). Results: The NBAS clusters’ predictive value was different at 3 d and at 4 wk. Orientation at 3 d and habituation at 4 wk were the best predictors of psychological problems. At 3 d, lower orientation scores and higher motor and habituation scores predicted higher scores in externalizing problems. Externalizing problems were least influenced by background variables. Only in girls were internalizing problems associated with lower orientation scores. At 4 wk, lower habituation cluster scores predicted higher scores in internalizing problems.

Conclusion: These findings suggest that the NBAS could be useful for identifying neonates at risk of later psychological problems.
Clarithromycin treatment and QT prolongation in childhood
Authors: GERMANAKIS IOANNIS , GALANAKIS EMMANOUIL , PARTHENAKIS FRAGISKOS , VARDAS PANOS E. , KALMANTI MARIA
Published: June 2006

The effect of clarithromycin on the QT interval was studied in a group of 28 children treated for respiratory tract infections. QTc was measured before and following 24 h of treatment. A modest (average 22 ms, 95% CI 14–30 ms) but significant QTc prolongation (p<0.001) was observed, with seven cases having a QTc >440 ms during treatment (including a single case with QTc >460 ms).

Conclusion: Serial QTc measurements are necessary for early detection of children at risk for drug‐induced arrhythmias.
The role of maternal depressed mood and behavioural soothing on infant response to routine vaccination
Authors: MOSCARDINO UGHETTA , AXIA GIOVANNA , ALTOÈ GIANMARCO
Published: June 2006

Background: It has been hypothesized that maternal characteristics may affect infants' experience of pain during stressful medical procedures. Aim: To investigate the role of maternal depressed mood on infants' response to vaccination, and to determine the effectiveness of different soothing behaviours in reducing infant distress. Methods: Twenty‐eight mothers and their healthy, full‐term infants participated in a prospective study. At infant age 2 mo, mothers completed the Edinburgh Postnatal Depression Scale (EPDS) and an adapted version of the Profile of Mood States (POMS); at infant age 4.5 mo, mothers were administered the mood scale and were observed with their infants during routine vaccination. Results: Higher levels of maternal depressed mood were predictive of a stronger infant pain response at routine vaccination. Contingencies derived from sequential analyses revealed that the soothing behaviours most effective in reducing infant distress were holding and face‐to‐face contact, whereas looking at the child from a distance was significantly associated with an increase in infant distress.

Conclusion: Our findings suggest that interventions to reduce infant pain during stressful medical procedures may include early detection and referral for maternal postpartum depression as well as the promotion of soothing behaviours involving close physical and emotional contact between mother and baby.
Is screening for vesicoureteral reflux mandatory in infants with antenatal renal pelvis dilatation?
Authors: LIDEFELT KARL‐JOHAN , EK SVERKER , MIHOCSA LAZLO
Published: June 2006

Aim: To determine whether postnatal ultrasound (US) can guide the use of voiding cystourethrography (VCUG) in infants with antenatally detected renal pelvis dilatation (ARPD). Methods: 14000 pregnant women consecutively underwent routine US examination during the second trimester. US examinations later in pregnancy were performed as follow‐up of previous anomalies or on obstetrical indications. One hundred and six fetuses with ARPD ≥5 mm were identified. Two postnatal US examinations were performed in the newborns: on the 5th to 7th day and during the 3rd week of life. The findings were considered normal when renal pelvis dilatation (RPD) was ≤7 mm on both US examinations, and no calyceal or ureteric dilatation or signs of renal dysplasia or other anomalies were present. VCUG was done 6 to 8 wk after birth. Results: In 53 of 103 analysable infants, the postnatal ultrasonographic findings were normal. The VCUG was abnormal in three of these 53 infants, all with vesicoureteral reflux (VUR) grade I. Of 50 infants who had abnormal US examinations, six had VUR, four of which were grade IV and V reflux.

Conclusion: In infants with ARPD who undergo two postnatal US examinations with RPD ≤7 mm and have no other abnormalities, VCUG is unnecessary.
Nectins and nectin‐like molecules: Roles in cell adhesion, polarization, movement, and proliferation
Authors: Ogita Hisakazu , Takai Yoshimi
Published: June 2006

Nectins and nectin‐like molecules (Necls) are immunoglobulin‐like cell adhesion molecules that constitute families containing four and five members, respectively. All members, except for Necl‐5, trans‐interact homophilically. Furthermore, all members, including Necl‐5, trans‐interact heterophilically with their respective specific partners among the members. Necl‐5 regulates cell movement and proliferation cooperatively with integrin αvβ3 and growth factor receptors. Nectins function as cell‐cell adhesion molecules at a variety of cell‐cell junctions, including adherens junctions, and regulate the initial step of cell‐cell junction formation. Nectins and integrin αvβ3 are further involved in the cross‐talk between cell‐matrix and cell‐cell junctions. Thus, both nectin and Necl family members play important roles in fundamental cellular functions, including cell adhesion, polarization, movement, and proliferation.
iubmb Life, 58: 334‐343, 2006
Membrane targeting and remodeling through phosphoinositide‐binding domains
Authors: Takenawa Tadaomi , Itoh Toshiki
Published: June 2006

In mammals, there are seven inositolphospholipids, collectively called phosphoinositides that serve as versatile molecules not only in receptor‐mediated signal transduction but also in a variety of cellular events such as cytoskeletal reorganization, membrane trafficking, cell proliferation and cell death. Recent studies have revealed that the latter functions are mediated by direct interactions between phosphoinositides and proteins. Such proteins contain two types of phosphoinositide‐binding regions; basic amino acid stretch and globular structural domain. Furthermore, spatially restricted compartment of phosphoinositides and their concentration are finely regulated by a large number of phosphoinositide kinases and ‐phosphatases, controlling localization‐specific metabolism of this simple lipid whose aberrations cause various diseases such as cancer and diabetes.
iubmb Life, 58: 296‐303, 2006
Multiple chaperone‐assisted formation of mammalian 20S proteasomes
Authors: Murata Shigeo
Published: June 2006

Protein degradation is essential for maintenance of cellular homeostasis. The majority of proteins are selectively degraded in eukaryotic cells by the ubiquitin‐proteasome system. The 26S proteasome selects target proteins that are covalently modified with polyubiquitin chains. The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. The catalytic activities are carried out by the core 20S proteasome. The eukaryotic 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, α1 ‐ 7β1 ‐ 7β1 ‐ 7α1 ‐ 7. Recent studies have revealed the mechanism responsible for the assembly of such a complex structure. This article recounts the observations that disclosed the biogenesis of 20S proteasomes and discusses the difference in the mechanism of assembly between archael, yeast, and mammalian 20S proteasomes.
iubmb Life, 58: 344‐348, 2006
Molecular mechanisms of intercellular communication in the hormonal and neural systems
Authors: Nakanishi Shigetada
Published: June 2006

This paper reviews our studies that have addressed the molecular mechanisms underlying the biosynthesis and reception of extracellular signaling molecules and integrative mechanisms of extracellular‐intracellular signaling transmission in biological systems. We introduced recombinant DNA technology into the neuroendocrine system and established the concept that a single peptide precursor encompasses multiple biologically active peptides and brings about coordinate functions in various biological systems. We then developed a novel functional cloning of membrane receptors and ion channels by combining an oocyte expression system with electrophysiology. We molecularly elucidated not only various peptide receptors, including the first demonstration of the molecular entity of a G protein‐coupled peptide receptor (GPCR), substance K receptor, and also diverse members of both G protein‐coupled metabotropic type and NMDA type of neurotransmitter glutamate receptors. We demonstrated many novel synaptic mechanisms involving distinct types of glutamate receptors in brain function and dysfunction. These include the mechanisms underlying segregation of light‐dark signals in visual transmission, discrimination and memory formation in olfactory transmission, and motor co‐ordination in the cerebellum, basal ganglia and the retinal network.
iubmb Life, 58: 349‐357, 2006
Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors
Authors: Boyer Laurent , Doye Anne , Rolando Monica , Flatau Gilles , Munro Patrick , Gounon Pierre , Clément René , Pulcini Céline , Popoff Michel R. , Mettouchi Amel , Landraud Luce , Dussurget Olivier , Lemichez Emmanuel
Published: June 2006

The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.
PI4P-signaling pathway for the synthesis of a nascent membrane structure in selective autophagy
Authors: Yamashita Shun-ichi , Oku Masahide , Wasada Yuko , Ano Yoshitaka , Sakai Yasuyoshi
Published: June 2006

Phosphoinositides regulate a wide range of cellular activities, including membrane trafficking and biogenesis, via interaction with various effector proteins that contain phosphoinositide binding motifs. We show that in the yeast Pichia pastoris, phosphatidylinositol 4′-monophosphate (PI4P) initiates de novo membrane synthesis that is required for peroxisome degradation by selective autophagy and that this PI4P signaling is modulated by an ergosterol-converting PpAtg26 (autophagy-related) protein harboring a novel PI4P binding GRAM (glucosyltransferase, Rab-like GTPase activators, and myotubularins) domain. A phosphatidylinositol-4-OH kinase, PpPik1, is the primary source of PI4P. PI4P concentrated in a protein–lipid nucleation complex recruits PpAtg26 through an interaction with the GRAM domain. Sterol conversion by PpAtg26 at the nucleation complex is necessary for elongation and maturation of the membrane structure. This study reveals the role of the PI4P-signaling pathway in selective autophagy, a process comprising multistep molecular events that lead to the de novo membrane formation.
Rac1 links integrin-mediated adhesion to the control of lactational differentiation in mammary epithelia
Authors: Akhtar Nasreen , Streuli Charles H.
Published: June 2006

The expression of tissue-specific genes during mammary gland differentiation relies on the coincidence of two distinct signaling events: the continued engagement of β1 integrins with the extracellular matrix (ECM) and a hormonal stimulus from prolactin (Prl). How the integrin and Prl receptor (PrlR) systems integrate to regulate milk protein gene synthesis is unknown. In this study, we identify Rac1 as a key link. Dominant-negative Rac1 prevents Prl-induced synthesis of the milk protein β-casein in primary mammary epithelial cells cultured as three-dimensional acini on basement membrane. Conversely, activated Rac1 rescues the defective β-casein synthesis that occurs under conditions not normally permissive for mammary differentiation, either in β1 integrin–null cells or in wild-type cells cultured on collagen. Rac1 is required downstream of integrins for activation of the PrlR/Stat5 signaling cascade. Cdc42 is also necessary for milk protein synthesis but functions via a distinct mechanism to Rac1. This study identifies the integration of signals provided by ECM and hormones as a novel role for Rho family guanosine triphosphatases.
Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement
Authors: Brangwynne Clifford P. , MacKintosh Frederick C. , Kumar Sanjay , Geisse Nicholas A. , Talbot Jennifer , Mahadevan L. , Parker Kevin K. , Ingber Donald E. , Weitz David A.
Published: June 2006

Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding only exceedingly small compressive forces. This discrepancy calls into question the structural role of microtubules, and highlights our lack of quantitative knowledge of the magnitude of the forces they experience and can withstand in living cells. We show that intracellular microtubules do bear large-scale compressive loads from a variety of physiological forces, but their buckling wavelength is reduced significantly because of mechanical coupling to the surrounding elastic cytoskeleton. We quantitatively explain this behavior, and show that this coupling dramatically increases the compressive forces that microtubules can sustain, suggesting they can make a more significant structural contribution to the mechanical behavior of the cell than previously thought possible.
A novel micro/nano 1 × 4 mechanical optical switch
Authors: Lin Wu-Lang , Fan Kuang-Chao , Chiang Li-Hung , Yang Yao-Joe , Kuo Wen-Cheng , Chung Tien-Tung
Published: June 2006

This paper presents the design, fabrication and testing of a novel 1 × 4 mechanical optical switch, whose components are fabricated by precision machining and MEMS technologies. The switch uses two relays as the two actuators whose switching direction is perpendicular to each other by an orthogonal arrangement. We adopt a direct fiber-to-fiber principle that aligns the input fiber directly to four output fibers. This configuration eliminates the use of traditional parts such as collimators, turning mirrors or prisms. In addition, due to the use of a fiber holder, the fiber position errors could be reduced to less than 0.27 µm using the two-stage geometry error reduction principle. We have successfully developed a simple and low-cost switch, which performs like most of the 1 × 4 mechanical optical switches that dominate the optics communications market. The advantages of our switch are a small size (20 × 20 × 25 mm3), low cost, high reliability, and the latching function does not need external force for maintaining the state. The experimental results showed that the insertion losses of the four channels are ch1: 0.68 dB, ch2: 1.49 dB, ch3: 0.71 dB and ch4: 0.97 dB. The switching time is 5 ms, the crosstalk ⩽80 dB. The reliability tests of the insertion loss after 10 000 cycles in four channels yield ch1: 1.67 dB, ch2: 1.63 dB, ch3: 0.75 dB and ch4: 0.98 dB. The size and the cost of our 1 × 4 mechanical optical switch are only about 1/5–1/10 and 1/10 of the series-connect-type and prism-type switches, respectively.
Set Relations in Social Research: Evaluating Their Consistency and Coverage
Authors: Ragin Charles C.
Published: June 2006

Because of its inherently asymmetric nature, set-theoretic analysis offers many interesting contrasts with analysis based on correlations. Until recently, however, social scientists have been slow to embrace set-theoretic approaches. The perception was that this type of analysis is restricted to primitive, binary variables and that it has little or no tolerance for error. With the advent of “fuzzy” sets and the recognition that even rough set-theoretic relations are relevant to theory, these old barriers have crumbled. This paper advances the set-theoretic approach by presenting simple descriptive measures that can be used to evaluate set-theoretic relationships, especially relations between fuzzy sets. The first measure, “consistency,” assesses the degree to which a subset relation has been approximated, whereas the second measure, “coverage,” assesses the empirical relevance of a consistent subset. This paper demonstrates further that set-theoretic coverage can be partitioned in a manner somewhat analogous to the partitioning of explained variation in multiple regression analysis.
Genomic organization of the sex-determining and adjacent regions of the sex chromosomes of medaka
Authors: Kondo Mariko , Hornung Ute , Nanda Indrajit , Imai Shuichiro , Sasaki Takashi , Shimizu Atsushi , Asakawa Shuichi , Hori Hiroshi , Schmid Michael , Shimizu Nobuyoshi , Schartl Manfred
Published: June 2006

Sequencing of the human Y chromosome has uncovered the peculiarities of the genomic organization of a heterogametic sex chromosome of old evolutionary age, and has led to many insights into the evolutionary changes that occurred during its long history. We have studied the genomic organization of the medaka fish Y chromosome, which is one of the youngest heterogametic sex chromosomes on which molecular data are available. The Y specific and adjacent regions were sequenced and compared to the X. The male sex-determining gene,dmrt1bY,appears to be the only functional gene in the Y-specific region. The Y-specific region itself is derived from the duplication of a 43-kb fragment from linkage group 9. All other coduplicated genes exceptdmrt1bYdegenerated. The Y-specific region has accumulated large stretches of repetitive sequences and duplicated pieces of DNA from elsewhere in the genome, thereby growing to 258 kb. Interestingly the non-recombining part of the Y did not spread out considerably from the original duplicated fragment, possibly because of a large sequence duplication bordering the Y-specific fragment. This may have conserved the more ancestral structure of the medaka Y and provides insights into some of the initial processes of Y chromosome evolution.
Ubiquitous selective constraints in the Drosophila genome revealed by a genome-wide interspecies comparison
Authors: Halligan Daniel L. , Keightley Peter D.
Published: June 2006

Non-coding DNA comprises ∼80% of the euchromatic portion of the Drosophila melanogaster genome. Non-coding sequences are known to contain functionally important elements controlling gene expression, but the proportion of sites that are selectively constrained is still largely unknown. We have compared the complete D. melanogaster and Drosophila simulans genome sequences to estimate mean selective constraint (the fraction of mutations that are eliminated by selection) in coding and non-coding DNA by standardizing to substitution rates in putatively unconstrained sequences. We show that constraint is positively correlated with intronic and intergenic sequence length and is generally remarkably strong in non-coding DNA, implying that more than half of all point mutations in the Drosophila genome are deleterious. This fraction is also likely to be an underestimate if many substitutions in non-coding DNA are adaptively driven to fixation. We also show that substitutions in long introns and intergenic sequences are clustered, such that there is an excess of substitutions <8 bp apart and a deficit farther apart. These results suggest that there are blocks of constrained nucleotides, presumably involved in gene expression control, that are concentrated in long non-coding sequences. Furthermore, we infer that there is more than three times as much functional non-coding DNA as protein-coding DNA in the Drosophila genome. Most deleterious mutations therefore occur in non-coding DNA, and these may make an important contribution to a wide variety of evolutionary processes.
Thousands of corresponding human and mouse genomic regions unalignable in primary sequence contain common RNA structure
Authors: Torarinsson Elfar , Sawera Milena , Havgaard Jakob H. , Fredholm Merete , Gorodkin Jan
Published: June 2006

Human and mouse genome sequences contain roughly 100,000 regions that are unalignable in primary sequence and neighbor corresponding alignable regions between both organisms. These pairs are generally assumed to be nonconserved, although the level of structural conservation between these has never been investigated. Owing to the limitations in computational methods, comparative genomics has been lacking the ability to compare such nonconserved sequence regions for conserved structural RNA elements. We have investigated the presence of structural RNA elements by conducting a local structural alignment, using FOLDALIGN, on a subset of these 100,000 corresponding regions and estimate that 1800 contain common RNA structures. Comparing our results with the recent mapping of transcribed fragments (transfrags) in human, we find that high-scoring candidates are twice as likely to be found in regions overlapped by transfrags than regions that are not overlapped by transfrags. To verify the coexpression between predicted candidates in human and mouse, we conducted expression studies by RT-PCR and Northern blotting on mouse candidates, which overlap with transfrags on human chromosome 20. RT-PCR results confirmed expression of 32 out of 36 candidates, whereas Northern blots confirmed four out of 12 candidates. Furthermore, many RT-PCR results indicate differential expression in different tissues. Hence, our findings suggest that there are corresponding regions between human and mouse, which contain expressed non-coding RNA sequences not alignable in primary sequence.
Molecular and Structural Characterization of Hexameric β-d-Glucosidases in Wheat and Rye
Authors: Sue Masayuki , Yamazaki Kana , Yajima Shunsuke , Nomura Taiji , Matsukawa Tetsuya , Iwamura Hajime , Miyamoto Toru
Published: June 2006

The wheat (Triticum aestivum) and rye (Secale cereale) β-d-glucosidases hydrolyze hydroxamic acid-glucose conjugates, exist as different types of isozyme, and function as oligomers. In this study, three cDNAs encoding β-d-glucosidases (TaGlu1a, TaGlu1b, and TaGlu1c) were isolated from young wheat shoots. Although the TaGlu1s share very high sequence homology, the mRNA level of Taglu1c was much lower than the other two genes in 48- and 96-h-old wheat shoots. The expression ratio of each gene was different between two wheat cultivars. Recombinant TaGlu1b expressed in Escherichia coli was electrophoretically distinct fromTaGlu1a and TaGlu1c. Furthermore, coexpression of TaGlu1a and TaGlu1b gave seven bands on a native-PAGE gel, indicating the formation of both homo- and heterohexamers. One distinctive property of the wheat and rye glucosidases is that they function as hexamers but lose activity when dissociated into smaller oligomers or monomers. The crystal structure of hexameric TaGlu1b was determined at a resolution of 1.8 Å. The N-terminal region was located at the dimer-dimer interface and plays a crucial role in hexamer formation. Mutational analyses revealed that the aromatic side chain at position 378, which is located at the entrance to the catalytic center, plays an important role in substrate binding. Additionally, serine-464 and leucine-465 of TaGlu1a were shown to be critical in the relative specificity for DIMBOA-glucose (2-O-β-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-glucose (7-demethoxy-DIMBOA-glucose).
The CLAVATA3/ESR Motif of CLAVATA3 Is Functionally Independent from the Nonconserved Flanking Sequences
Authors: Fiers Martijn , Golemiec Elzbieta , van der Schors Roel , van der Geest Lonneke , Li Ka Wan , Stiekema Willem J. , Liu Chun-Ming
Published: June 2006

It is believed that CLAVATA3 (CLV3) encodes a peptide ligand that interacts with the CLV1/CLV2 receptor complex to limit the number of stem cells in the shoot apical meristem of Arabidopsis thaliana; however, the exact composition of the functional CLV3 product remains a mystery. A recent study on CLV3 shows that the CLV3/ESR (CLE) motif, together with the adjacent C-terminal sequence, is sufficient to execute CLV3 function when fused behind an N-terminal sequence of ERECTA. Here we show that most of the sequences flanking the CLE motif of CLV3 can be deleted without affecting CLV3 function. Using a liquid culture assay, we demonstrate that CLV3p, a synthetic peptide corresponding to the CLE motif of CLV3, is able to restrict the size of the shoot apical meristem in clv3 seedlings but not in clv1 seedlings. In accordance with this decrease in meristem size, application of CLV3p to in vitro-grown clv3 seedlings restricts the expression of the stem cell-promoting transcription factor WUSCHEL. Thus, we propose that the CLE motif is the functional region of CLV3 and that this region acts independently of its adjacent sequences.
The BAM1/BAM2 Receptor-Like Kinases Are Important Regulators ofArabidopsisEarly Anther Development
Authors: Hord Carey L.H. , Chen Changbin , DeYoung Brody J. , Clark Steven E. , Ma Hong
Published: June 2006

Anther development involves the formation of several adjacent cell types required for normal male fertility. Only a few genes are known to be involved in early anther development, particularly in the establishment of these different cell layers. Arabidopsis thaliana  BAM1 (for BARELY ANY MERISTEM) and BAM2 encode CLAVATA1-related Leu-rich repeat receptor-like kinases that appear to have redundant or overlapping functions. We characterized anther development in the bam1 bam2 flowers and found that bam1 bam2 anthers appear to be abnormal at a very early stage and lack the endothecium, middle, and tapetum layers. Analyses using molecular markers and cytological techniques of bam1 bam2 anthers revealed that cells interior to the epidermis acquire some characteristics of pollen mother cells (PMCs), suggesting defects in cell fate specification. The pollen mother-like cells degenerate before the completion of meiosis, suggesting that these cells are defective. In addition, the BAM1 and BAM2 expression pattern supports both an early role in promoting somatic cell fates and a subsequent function in the PMCs. Therefore, analysis of BAM1 and BAM2 revealed a cell–cell communication process important for early anther development, including aspects of cell division and differentiation. This finding may have implications for the evolution of multiple signaling pathways in specifying cell types for microsporogenesis.
TheArabidopsisSUPPRESSOR OF AUXIN RESISTANCE Proteins Are Nucleoporins with an Important Role in Hormone Signaling and Development
Authors: Parry Geraint , Ward Sally , Cernac Alex , Dharmasiri Sunethra , Estelle Mark
Published: June 2006

Nucleocytoplasmic transport of macromolecules is regulated by a large multisubunit complex called the nuclear pore complex (NPC). Although this complex is well characterized in animals and fungi, there is relatively little information on the NPC in plants. The suppressor of auxin resistance1 (sar1) and sar3 mutants were identified as suppressors of the auxin-resistant1 (axr1) mutant. Molecular characterization of these genes reveals that they encode proteins with similarity to vertebrate nucleoporins, subunits of the NPC. Furthermore, a SAR3–green fluorescent protein fusion protein localizes to the nuclear membrane, indicating that SAR1 and SAR3 are Arabidopsis thaliana nucleoporins. Plants deficient in either protein exhibit pleiotropic growth defects that are further accentuated in sar1 sar3 double mutants. Both sar1 and sar3 mutations affect the localization of the transcriptional repressor AXR3/INDOLE ACETIC ACID17, providing a likely explanation for suppression of the phenotype conferred by axr1. In addition, sar1 sar3 plants accumulate polyadenylated RNA within the nucleus, indicating that SAR1 and SAR3 are required for mRNA export. Our results demonstrate the important role of the plant NPC in hormone signaling and development.
STABILIZED1, a Stress-Upregulated Nuclear Protein, Is Required for Pre-mRNA Splicing, mRNA Turnover, and Stress Tolerance in Arabidopsis
Authors: Lee Byeong-ha , Kapoor Avnish , Zhu Jianhua , Zhu Jian-Kang
Published: June 2006

In plants, many gene transcripts are very unstable, which is important for the tight control of their temporal and spatial expression patterns. To identify cellular factors controlling the stability of unstable mRNAs in plants, we used luciferase imaging in Arabidopsis thaliana to isolate a recessive mutant, stabilized1-1 (sta1-1), with enhanced stability of the normally unstable luciferase transcript. The sta1-1 mutation also causes the stabilization of some endogenous gene transcripts and has a range of developmental and stress response phenotypes. STA1 encodes a nuclear protein similar to the human U5 small ribonucleoprotein–associated 102-kD protein and to the yeast pre-mRNA splicing factors Prp1p and Prp6p. STA1 expression is upregulated by cold stress, and the sta1-1 mutant is defective in the splicing of the cold-induced COR15A gene. Our results show that STA1 is a pre-mRNA splicing factor required not only for splicing but also for the turnover of unstable transcripts and that it has an important role in plant responses to abiotic stresses.
X‐ray structure of potato epoxide hydrolase sheds light on substrate specificity in plant enzymes
Authors: Mowbray Sherry L. , Elfström Lisa T. , Ahlgren Kerstin M. , Andersson C. Evalena , Widersten Mikael
Published: June 2006

Epoxide hydrolases catalyze the conversion of epoxides to diols. The known functions of such enzymes include detoxification of xenobiotics, drug metabolism, synthesis of signaling compounds, and intermediary metabolism. In plants, epoxide hydrolases are thought to participate in general defense systems. In the present study, we report the first structure of a plant epoxide hydrolase, one of the four homologous enzymes found in potato. The structure was solved by molecular replacement and refined to a resolution of 1.95 Å. Analysis of the structure allows a better understanding of the observed substrate specificities and activity. Further, comparisons with mammalian and fungal epoxide hydrolase structures reported earlier show the basis of differing substrate specificities in the various epoxide hydrolase subfamilies. Most plant enzymes, like the potato epoxide hydrolase, are expected to be monomers with a preference for substrates with long lipid‐like substituents of the epoxide ring. The significance of these results in the context of biological roles and industrial applications is discussed.
Enhancement of bound‐state residual dipolar couplings: Conformational analysis of lactose bound to Galectin‐3
Authors: Zhuang Tiandi , Leffler Hakon , Prestegard James H.
Published: June 2006

Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long‐range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements dominated by bound contributions, RDCs of exchanging ligands can be dominated by free‐state contributions. Here we present a strategy for enhancement of RDCs from bound states that is based on specifically enhancing the alignment of the protein to which a ligand will bind. The protein is modified by addition of a hydrophobic alkyl tail that anchors it to the bicelles that are a part of the ordering medium needed for RDC measurement. As an illustration, we have added a propyl chain to the C terminus of the carbohydrate recognition domain of the protein, Galectin‐3, and report enhanced RDCs that prove consistent with known bound‐ligand geometries for this protein.
The effects of sodium sulfate, glycosaminoglycans, and Congo red on the structure, stability, and amyloid formation of an immunoglobulin light‐chain protein
Authors: Mclaughlin Richard W. , De Stigter Janelle K. , Sikkink Laura A. , Baden Elizabeth M. , Ramirez‐Alvarado Marina
Published: June 2006

Light‐chain amyloidosis (AL) is characterized by immunoglobulin light‐chain fragments aggregating into amyloid fibrils that deposit extracellularly in vital organs such as the kidney, the heart, and the liver, resulting in tissue degeneration and organ failure, leading to death. Cardiac involvement is found in 50% of AL patients and presents the most severe cases with a life expectancy of less than a year after diagnosis. In this study, we have characterized the variable domain of a cardiac AL patient light chain called AL‐09. AL‐09 folds as a β‐sheet and is capable of forming amyloid fibrils both in the presence of sodium sulfate and in self‐seeded reactions under physiological conditions. Glycosaminoglycans such as dermatan sulfate and heparin promote amyloid formation of self‐seeded AL‐09 reactions, while the glycosaminoglycan chondroitin sulfate A stabilized oligomeric intermediates and did not elongate the preformed fibrils (nucleus) present in the reaction. Finally, the histological dye Congo red, known to bind to the cross β‐sheet structure of amyloid fibrils, inhibits AL‐09 amyloid fibril formation in the presence of sodium sulfate and in self‐seeded reactions. This paper provides insight into the impact of different reagents on light‐chain stability, structure, amyloid fibril formation, and inhibition.
A composite score for predicting errors in protein structure models
Authors: Eramian David , Shen Min‐yi , Devos Damien , Melo Francisco , Sali Andrej , Marti‐Renom Marc A.
Published: June 2006

Reliable prediction of model accuracy is an important unsolved problem in protein structure modeling. To address this problem, we studied 24 individual assessment scores, including physics‐based energy functions, statistical potentials, and machine learning–based scoring functions. Individual scores were also used to construct ∼85,000 composite scoring functions using support vector machine (SVM) regression. The scores were tested for their abilities to identify the most native‐like models from a set of 6000 comparative models of 20 representative protein structures. Each of the 20 targets was modeled using a template of <30% sequence identity, corresponding to challenging comparative modeling cases. The best SVM score outperformed all individual scores by decreasing the average RMSD difference between the model identified as the best of the set and the model with the lowest RMSD (ΔRMSD) from 0.63 Å to 0.45 Å, while having a higher Pearson correlation coefficient to RMSD (r = 0.87) than any other tested score. The most accurate score is based on a combination of the DOPE non‐hydrogen atom statistical potential; surface, contact, and combined statistical potentials from MODPIPE; and two PSIPRED/DSSP scores. It was implemented in the SVMod program, which can now be applied to select the final model in various modeling problems, including fold assignment, target–template alignment, and loop modeling.
Mutagenic exploration of the cross‐seeding and fibrillation propensity of Alzheimer's β‐amyloid peptide variants
Authors: Peim Alexander , Hortschansky Peter , Christopeit Tony , Schroeckh Volker , Richter Walter , Fändrich Marcus
Published: June 2006

Amyloid formation is a nucleation‐dependent process that is accelerated dramatically in vivo and in vitro upon addition of appropriate fibril seeds. A potent species barrier can be effective in this reaction if donor and recipient come from different biological species. This species barrier is thought to reflect differences in the amino acid sequence between seed and target polypeptide. Here we present an in vitro mutagenic cross‐seeding analysis of Alzheimer's Aβ(1‐40) peptide in which we mapped out the effect of systematically varied amino acid replacements on the propensity of seed‐dependent amyloid fibril formation. We find that the susceptibility of different peptides toward cross‐seeding relates to the intrinsic aggregation propensity of the respective polypeptide chain and, therefore, to properties such as β‐sheet propensity and hydrophobicity. These data imply that the seed‐dependent formation of amyloid‐like fibrils is affected by the intrinsic properties of the polypeptide chain in a manner that is similar to what has been described previously for aggregation reactions in general. Hence, the nucleus acts in this case as a catalyst that promotes the fibrillation of different polypeptide chains according to their intrinsic structural predilection.
Native state energetics of the Src SH2 domain: Evidence for a partially structured state in the denatured ensemble
Authors: Wildes David , Anderson L. Meadow , Sabogal Alex , Marqusee Susan
Published: June 2006

We have defined the free‐energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two‐state mechanism, without populating any intermediates. Native state hydrogen–deuterium exchange confirms this two‐state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native‐state proteolysis demonstrates that this “super protection” does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent ∼30‐fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured‐state ensemble.
Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy
Authors: Modrak‐Wójcik Anna , Stępniak Katarzyna , Akoev Vladimir , Żółkiewski Michał , Bzowska Agnieszka
Published: June 2006

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150 ± 10 kDa, regardless of the conditions investigated—protein concentration, 0.18–1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4–20°C. The parameters obtained from the self‐associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 μg/mL–1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s020,w = 7.7 ± 0.3 and 8.3 ± 0.4 S, respectively. The molecular mass of the sedimenting particle (146 ± 30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single‐step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.
4. Robust Spatial Analysis of Rare Crimes: An Information-Theoretic Approach
Authors: Bhati Avinash Singh
Published: June 2006

In this paper we describe a semiparametric information-theoretic framework for modeling the determinants of rare events aggregated at intracity areal units while allowing for various forms of error correlation structures. The approach is applied to an examination of the effects of socioeconomic and demographic macro-characteristics of communities on the amount of violence they experience. We investigate and find evidence of some instability in these processes across types of violence and level of areal aggregation. However, we also find evidence of a stable predictor—resource deprivation—for all the types of violence analyzed and at both levels of areal aggregation considered. In addition, we find evidence of a spillover effect of a community's resource deprivation on the level of violence its neighboring areas can expect. We discuss our findings in light of their substantive, methodological, and practical implications.