bluuebunny/crossref_metadata_2025 huggingface dataset:
300 GLUCOSAMINE SUPPLEMENTATION DURING IN VITRO MATURATION LEADS TO PERTURBED DEVELOPMENTAL CAPACITY OF BOVINE CUMULUS OOCYTE COMPLEXES
Authors: Sutton-McDowall M. , Gilchrist R. , Thompson J.
Published: December 2004

Glucose is a primary energy substrate required for successful in vitro oocyte maturation (IVM). However, most maturation media contain more glucose than that seen in follicular fluid (2.3 mM vs. 5.6 mM in TCM199). Glucosamine (Glc) as an alternative substrate for extracellular matrix during cumulus expansion reduced glucose uptake by bovine cumulus oocyte complexes (COCs, Sutton-McDowall et al. 2004 Reproduction 128, 313–319). As this could enable a reduction in glucose concentrations to physiological levels in IVM medium of COCs, the aim of this study was to investigate the influence of Glc supplementation on oocyte developmental capacity. Bovine COCs were matured in synthetic follicular fluid medium (SFFM, a defined medium based on the composition of follicular fluid, plus 5.6 mM glucose, FSH, hCG and BSA, Sutton-McDowall et al. 2004 Reprod. Fertil. Dev. 16 sup, 204) ± 5 mM Glc. After 24 h, either nuclear maturation (rep = 8, n = 160) or blastocyst development 8 days post-fertilization (rep = 5, n = 400) was determined. Data was arcsine transformed and analyzed by ANOVA, followed by Tukey's test. While the presence of Glc did not affect the completion of nuclear maturation and early cleavage, +Glc led to severely perturbed blastocyst development (−Glc, 32.5 ± 1.9% vs. +Glc, 4.7 ± 3.9%, P < 0.001). Glc supplementation in somatic cells is well-known to down-regulate the phosphatidylinositol-3-kinase (PI3K) signalling pathway, reducing protein synthesis and other cell survival mechanisms. Therefore, oocyte protein synthesis (measured by [2,3,4,5,6-3H] phenylalanine incorporation, rep = 5, n = 200) and embryo development (rep = 6, n = 720) following IVM in SFFM ± Glc ± EGF (a PI3K pathway stimulator) was determined. Glc supplementation led to a 40% decrease in protein synthesis compared to −Glc, while the combination of +Glc + EGF significantly increased protein synthesis by 60%. However, IVM + EGF + Glc did not improve blastocyst rates (main effect: −Glc 41.6 ± 6.6% vs. +Glc, 6.6 ± 1.7%, P < 0.001). Additionally, COCs were also cultured in SFFM ± 50 μM LY294002 (a specific PI3K inhibitor) and nuclear maturation (rep = 5, n = 200) or blastocyst development 8 days post-fertilization (rep = 4, n = 200) was determined. Despite the presence of LY294002 leading to 43% less COCs completing nuclear maturation (P < 0.001), blastocyst development was not affected (mean = 38.8 ± 3.2%). These results demonstrate that Glc supplementation during IVM has no effect on nuclear maturation or early development but is detrimental to oocyte developmental capacity by severely perturbing blastocyst development. However, the diminished developmental capacity appears to be independent of the well-characterized Glc down-regulation of the PI3K signalling pathway.
This work was supported by the Australian Research Council (SPIRT, C00107702) and Cook Australia Pty Ltd.
322 DIFFERENTIAL DEVELOPMENT OF RABBIT EMBRYOS FOLLOWING MICROINSEMINATION USING SPERM AND SPERMATIDS
Authors: Ogonuki N. , Inoue K. , Miki H. , Hirose Y. , Okada H. , Shimozawa N. , Takeiri S. , Nagashima H. , Sankai T. , Ogura A.
Published: December 2004

Microinsemination is a technique that delivers male germ cells directly into the ooplasm. The efficiency of fertilization and subsequent embryo development after microinsemination varies with species and the male germ cells used. This study examined the developmental ability of rabbit embryos in vitro and in vivo following microinsemination using haploid male germ cells at different stages. First, we injected rabbit spermatozoa, elongated spermatids, and round spermatids into mouse oocytes to assess their oocyte-activating capacity. Mouse oocytes are a good experimental model for assessing the oocyte-activating capacity of male germ cells from different species. The majority of mouse oocytes were activated irrespective of the stage of rabbit male germ cells injected (77, 61, and 73% for spermatozoa, elongated spermatids, and round spermatids, respectively). By contrast, these male germ cells activated homologous rabbit oocytes at rates of 100, 59, and 29%, respectively. After 120 h in culture, 69, 55, and 13% of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts, respectively. The rate of embryo development into blastocysts following round spermatid injection was significantly improved when oocytes were activated by an electric pulse shortly before microinsemination. The total number of cells was counted in embryos that reached the morula/blastocyst stages in culture using nuclear-staining with propidium iodide. The average cell number of embryos derived from elongated (89 ± 41; mean ± SD) or round spermatid (98 ± 34) injection was significantly lower than that of control embryos (in vivo fertilization) (211 ± 44) (P < 0.01). After 24 h in culture, some four- to eight-cell-stage embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from embryos involving sperm (4 offspring/16 transfers; 25%) and elongated spermatid (3/26; 12%) injection, but none from those involving round spermatid injection (0/68). These findings indicate that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. Immaturity of the nuclear genome or difficulty in coordinating the behavior of the male and female chromosomes might compromise embryo development.
331 MODIFIED SINGLE-STRANDED OLIGONUCLEOTIDE-RECOMBINASE COMPLEX MEDIATES GENE TARGETING IN MOUSE EMBRYOS
Authors: Kang J.H. , Won J.Y. , Shim H.
Published: December 2004

Gene targeting is an in situ manipulation of an endogenous gene in a precise manner by the introduction of exogenous DNA. The process of gene targeting involves a homologous recombination reaction between the targeted genomic sequence and an exogenous targeting vector. In elucidating the function of many genes, gene targeting has become the most important method of choice. Conventional gene targeting has been achieved through the use of embryonic stem cells. However, such a procedure is often long, tedious, and expensive and has been limited in the mouse only due to a lack of usable embryonic stem cells in other species. This study was carried out to develop a much simplified procedure of gene targeting using E. coli recombinase recA and modified single-stranded oligonucleotides. The new procedure was attempted to modify X-linked hypoxanthine phosphoribosyltransferase (HPRT) gene. The single-stranded oligonucleotide to target exon 3 of HPRT was 74 bases in length and included three phosphorothioate linkages at each terminus (also known as S-oligo) so as to be resistant against exonucleases when introduced into zygotes. The oligonucleotide sequence was homologous to the target gene except for a single nucleotide that induces a mismatch between the introduced oligonucleotide and endogenous HPRT gene. Although the exact mechanism is yet unknown, endogenous repairing of such a mismatch would give rise to the conversion of TAT to TAG stop codon, thereby losing the function of the target gene. Prior to an introduction into zygotes, modified single-stranded oligonucleotides were preincubated with recA recombinase to enhance the homologous recombination. The recA-oligonucleotide complex was microinjected into the pronuclei of zygotes. Individual microinjected embryos that developed to the blastocyst stage were analyzed for the expected nucleotide conversion using PCR and subsequent sequencing. The conversion of TAT to TAG stop codon was confirmed in two embryos among forty tested blastocysts, so that the frequency of gene targeting was approximately 5%. The result suggests that the gene targeting was feasible by this relatively easier direct method. Subsequent transfer of gene-targeted embryos to recipients to obtain transgenic mice missing the function of HPRT gene is underway. Further technical refinement and enhancement of homologous recombination frequency will be required for the practical use of this new approach for gene targeting in mice.
335 A PROCEDURE COMBINING ISTAT® ANALYSIS WITH OPU TO STUDY BOVINE FOLLICULAR ENVIRONMENTS
Authors: Berg M.C. , Beaumont S.E. , Peterson A.J. , Berg D.K.
Published: December 2004

Our previous work successfully analysed follicular fluid (45/61, 74% sampling success) from live cows using a portable clinical gas tension analyser (iSTAT Corp., Princeton, NJ). However, the oocyte recovery rate (7/24, 29%) from preovulatory follicles (POF) was disappointingly low, perhaps due to a need for the second puncture to aspirate the remaining follicular contents. This work describes a modified procedure that allowed a follicular fluid (FF) sample to be collected and stored in a gas-impermeable manner and the remaining follicular contents to be directly aspirated with or without follicular flushing from a single puncture. This modified dual port slip-luer hub, designed for use with a disposable needle ovum pick-up (OPU) system (PieMed, Netherlands), used a low negative pressure (30 mmHg), medium flow rate (25 mL/min) aspiration system with syringe flushing. FF samples for analysis were collected and stored in glass capillary tubes and the rest of the follicular contents/flushings were collected into tubes containing 10 mL collection medium and searched for oocytes. The hub's larger port, with a fitted Y connector, allows an initial flow of FF to be drawn into a capillary tube which has a polyvinylchloride (PVC) powder plugged end. When the PVC powder is permeated with FF it forms a seal effectively capping the tube. The remaining FF is diverted down the other leg of the Y connector using negative pressure. Flushing medium can be injected through the smaller port into the follicle while negative pressure is interrupted. A pilot trial examined gas tension, pH and ion concentrations from POF, dominant (D), and hormonally stimulated (S) follicles. Twenty synchronised Friesian cattle were scanned daily and follicles were tracked and mapped. The POF and D follicles were sampled on Day 20 or Day 10 respectively of the estrous cycle. The cows were then subjected to four weekly stimulations (used CIDR-B device inserted Day 2 and removed Day 5, 120 mg NIH-FSH-P1 administered twice daily on Day 5 and 6). Follicles were sampled and aspirated on Day 7. Successful FF analysis was achieved in 88% (110/125) of follicles attempted while oocyte recovery rates from POF, D, and S follicles were 56% (9/16), 54% (6/11) and 43% (36/83) respectively, showing it is possible to combine portable gas tension analysis with effective OPU. However, further work is needed to elucidate the correlation between the follicular environment and the oocyte developmental competence.

Table 1.
Follicular fluid sample analysis
72 EFFECT OF CELL CYCLE PHASE OF GENE-MANIPULATED FETAL FIBROBLASTS ON THE DEVELOPMENT OF CLONED BOVINE EMBRYOS
Authors: Urakawa M. , Sawada T. , Sendai Y. , Shinkai Y. , Ideta A. , Kubota K. , Hoshi H. , Aoyagi Y.
Published: December 2004

Transgenic bovine fetuses and offspring can be produced by using gene-modified somatic cells and clones of these cells. In this study, we examined the effects of specific cell cycle (early G1 phase) of donor cell (gene-manipulated fibroblasts) on the development of the nuclear transfer (NT) embryos into blastocysts and on the fetus production after embryo transfer. The gene-manipulated (tg; targeting of one or both alleles of gene encoding α-1,3-galactosyltransferase) or non-manipulated (control) bovine fetal fibroblasts were used for NT. The fibroblasts transfected with the targeting vector were selected with 0.4 mg mL−1 G418. The G418-resistant cells were monitored by PCR and Southern blot analysis. The cells (tg cells) in which homologous recombination occurred were used for NT. For NT, both tg cells and control cells were cultured in DMEM with 10% FCS. Early G1 cells were prepared by choosing pairs of bridged cells derived from mitotic phase cells (Urakawa M et al. 2004 Theriogenology 62, 714–728), and non-synchronized cells were obtained from a culture plate that had reached 60–80% confluence. Each donor cell was inserted into an enucleated, in vitro-matured (19 h) oocyte. Oocyte-cell couples were electrofused and activated with calcium ionophore and cycloheximide. The NT embryos were then co-cultured with bovine oviduct epithelial cells in CR1aa with 5% CS. The blastocyst rates were determined at 6 days after NT. The blastocysts were nonsurgically transferred to recipient heifers, and the developmental rate to the normal fetus was examined by the recovery of fetus or by using ultrasonography at Days 35–42. Data were analyzed by ANOVA. The developmental rate to the blastocyst stage did not differ significantly between tg (28.4%, 128/425) and control (25.4%, 181/739) cell groups. In the control group, the blastocyst rate of embryos constructed from early G1 phase fibroblasts (25.7%, 80/311) was not significantly different from that of embryos constructed with non-synchronized fibroblasts (23.6%, 101/428). In contrast, the blastocyst rate of tg cell derived-embryos was lower (P < 0.05) in early G1 phase (23.5%, 71/302) than in non-synchronized cell phase (46.3%, 57/123). The rate of development to a normal fetus in the tg group (15.4%, 4/26) was significant lower than that in the control group (62.5%, 25/40). For both the tg group and the control group, the rate of development to fetus tended to be higher (P > 0.05) for blastocysts derived from cells at the early G1 phase than for blastocysts derived from non-synchronized cells (tg group, 25.0%, 3/12 v. 7.1%, 1/14; control group, 90.0%, 9/10 v. 53.3%, 16/30). These results demonstrate that gene modification of fetal fibroblasts affects the development of NT embryos to fetuses. In addition, the synchronization of genetically modified donor cells to the early G1 phase may increase the potential to develop to a normal fetus.
9 DOES STRAW CONCENTRATION AFFECT POST-THAW SPERM QUALITY IN AI BULL SIRES?
Authors: Ballester J. , Johannisson A. , Håård M. , Gustafsson H. , Rodriguez-Martinez H.
Published: December 2004

There is increasing interest in decreasing the number of spermatozoa per AI dose, owing to the discrimination of threshold concentration and fertility, economical revenues and the use of sexed semen. This study evaluated the quality (as sperm viability, acrosome integrity, membrane stability and chromatin stability) post-thaw of semen collected from four elite AI sires and frozen at 15 × 106 (control) or 2 × 106 spermatozoa (spz) per dose to disclose eventual deleterious extension effects. Semen collected via a.v. from 4 élite AI sires was split-processed and frozen in 0.25 mL plastic straws under commercial conditions, at either 2 × 106 or 15 × 106 spz/straw (the latter as control). The semen parameters were within acceptable limits of normality. The post-thaw samples showed acceptable motility (above 50%). Sperm viability and stability were assessed post-thaw using flow cytometry of cells loaded with SYBR-14 and propidium iodide (PI) for sperm viability (membrane integrity), and with carboxy-SNARF-1 (SNARF; Invitrogen AB, Frolunda, Sweden), PI, and FITC-Pisum sativum agglutinin (PSA, triple stain) for acrosome status. Membrane stability status was measured with Annexin-V/PI, while sperm chromatin condensation and stability were assessed following in situ acid-induced DNA denaturation and staining with acridine orange. No significant differences were seen between the two concentrations regarding sperm motility, plasma membrane integrity (SYBR/PI, SNARF), or chromatin stability. The highly extended semen (2 × 106 spz/straw), however, showed a higher (P < 0.05) frequency of spermatozoa with translocated phosphatydil serine (as detected with Annexin-V), indicating their plasma membranes had become unstable. Also, there were more spermatozoa showing acrosomal damage (PSA). In conclusion, low concentrations of spermatozoa do not properly sustain conventional cryopreservation, although damages appear restricted to the sperm membrane. These changes in the stability of the plasma membrane apparently did not affect the viability of the spermatozoa, although they may negatively affect their lifespan. These findings may have an impact on the care that must be taken when cryopreserving low concentrations of spermatozoa with conventional freezing protocols.
This work was supported by the Swedish Farmers’ Foundation for Research in Agriculture (SLF) and FORMAS, Stockholm.
99 COMPARISON OF IN VITRO DEVELOPMENT FOLLOWING CRYOPRESERVATION OF MEISHAN AND WHITE CROSS SWINE EMBRYOS
Authors: White B. , Montagner M. , Mills G. , Gonçalves P. , Christenson R.
Published: December 2004

Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253–260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5- to 10-μL microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN2) for 15 s and then plunged into LN2. The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN2-submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2.5% EG + 0.29 M sucrose; (3) BECM + 0.3 M sucrose; and (4) BECM alone. All procedures were performed with solutions maintained at 37°C. Cryopreserved and control embryos were cultured in 50 μL drops of modified Whitten's medium + 1.5% BSA under oil at 37°C in a 5% CO2 in air environment and scored daily for development. For embryos undergoing cryopreservation, retrieval rates from cryovials were 92% and 96% for M and WC, respectively. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than for WC (44%; P < 0.001; χ2-test). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than for frozen embryos from both breeds. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98% and 95%, respectively), but more M embryos developed to the hatched blastocyststage (22% for M v. 9% for WC; P < 0.05). Our results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos.
Funding for M. Montagner was provided by CAPES, Brazil.
Relative efficiency of rhizobacteria for auxin biosynthesis in rhizosphere and non-rhizosphere soils
Authors: Khalid Azeem , Tahir Shermeen , Arshad Muhammad , Zahir Zahir Ahmad
Published: December 2004

Biosynthesis of auxins in the rhizosphere of different crops may vary because of quantitative and qualitative variations in microbial population and root exudation. A laboratory study was conducted to assess in vitro auxin biosynthesis, and biosynthesis in rhizosphere and non-rhizosphere soils of different crops (maize, sorghum, mungbean, cotton). Soils were inoculated with selected rhizobacteria with and without the auxin precursor L-tryptophan (L-TRP). Auxins were detected by colourimetry as indole acetic acid equivalents and confirmed by high performance liquid chromatography. Results revealed that 83% of the 60 rhizobacteria were capable of producing auxins in the absence of L-TRP. Auxin biosynthesis by the 8 most efficient rhizobacteria ranged from 5.0 to 12.1 mg/L broth medium. A comparison of rhizosphere v. non-rhizosphere soils indicated a greater accumulation of auxins in the rhizosphere soils than non-rhizosphere soils. Overall, inoculation of rhizosphere soils with selected rhizobacteria resulted in greater production of auxin (up to 10.4 mg/kg soil) than in inoculated non-rhizosphere soils (up to 5.76 mg/kg). Moreover, efficiency of these rhizobacteria for auxin biosynthesis in both rhizosphere and non-rhizosphere soils differed with crop and bacterial strain. Some rhizobacterial strains exhibited superiority over the indigenous microflora for auxin biosynthesis in soil. Application of L-TRP promoted auxin biosynthesis in both rhizosphere and non-rhizosphere soils. These findings imply that inoculation with suitable strains and/or amendment with L-TRP could promote auxin synthesis in the rhizosphere soil of a given crop, which may have consequences for better plant/crop growth.
Calibration of the RothC model to turnover of soil carbon under eucalypts and pines
Authors: Paul K. I. , Polglase P. J.
Published: December 2004

Abstract
The FullCAM model was developed for full carbon accounting in agriculture and forests at project and national scales. For forest systems, FullCAM links the empirical CAMFor model to models of tree growth (3PG), litter decomposition (GENDEC), and soil carbon turnover (RothC). Our objective was to calibrate RothC within the FullCAM framework using 2 long-term forestry experiments where productivity had been manipulated and archived and new soil samples were available for analysis of carbon within the various pools described by RothC. Inputs of carbon to soil at these trials were estimated by calibrating FullCAM to temporal data on above-ground growth, litterfall, and accumulation of litter. Two alternative submodels are available in FullCAM (CAMFor and GENDEC) for predicting decomposition of litter, and thus the input of carbon into the soil. Calibration of RothC was most sensitive to the partitioning of carbon during decomposition of debris between that lost as CO2 and that transferred to soil. Turnover of soil carbon was best simulated when the proportion of carbon lost to CO2 from relatively labile pools of debris was 77% (when simulated by CAMFor) and 95% (when simulated by GENDEC), whereas resistant pools of debris lost about 40% to CO2 during decomposition. Although rates of decomposition of pools of soil carbon were originally developed in RothC for agricultural soils, these constants were found to be also suitable for soils under plantation systems.
Gypsum solubility in seawater, and its application to bauxite residue amelioration
Authors: Kopittke P. M. , Menzies N. W. , Fulton I. M.
Published: December 2004

The solubilities and dissolution rates of 3 gypsum sources [analytical grade reagent (AR), phosphogypsum (PG), mined gypsum (MG)] with 6 MG size fractions (>2.0, 1.0–2.0, 0.5–1.0, 0.25–0.5, 0.125–0.25, <0.125 mm) were investigated in triple-deionised water (TDI) and seawater to examine their suitability for bauxite residue amelioration. Gypsum solubility was greater in seawater (3.8 g/L) than TDI (2.9 g/L) due to the ionic strength effect, with dissolution in both TDI and seawater following first-order kinetics. Dissolution rate constants varied with gypsum source (AR > PG > MG) due to reactivity and surface area differences, with 1 : 20 gypsum : solution suspensions reaching saturation within 15 s (AR) to 30 min (MG >2.0 mm). The ability of bauxite residue to adsorb Ca from solution was also examined. The quantity of the total solution Ca adsorbed was found to be small (5%). These low rates of solution Ca adsorption, combined with the comparatively rapid dissolution rates, preclude the application of gypsum to the residue sand/seawater slurry as a method for residue amelioration. Instead, direct field application to the residue would ensure more efficient gypsum use. In addition, the formation of a sparingly soluble CaCO3 coating around the gypsum particles after mixing in a highly alkaline seawater/supernatant liquor solution greatly reduced the rate of gypsum dissolution.
Fire weather index system components for large fires in the Canadian boreal forest
Authors: Amiro B. D. , Logan K. A. , Wotton B. M. , Flannigan M. D. , Todd J. B. , Stocks B. J. , Martell D. L.
Published: December 2004

Canadian Fire Weather Index (FWI) System components and head fire intensities were calculated for fires greater than 2 km2 in size for the boreal and taiga ecozones of Canada from 1959 to 1999. The highest noon-hour values were analysed that occurred during the first 21 days of each of 9333 fires. Depending on ecozone, the means of the FWI System parameters ranged from: fine fuel moisture code (FFMC), 90 to 92 (82 to 96 for individual fires); duff moisture code (DMC), 38 to 78 (10 to 140 for individual fires); drought code (DC), 210 to 372 (50 to 600 for individual fires); and fire weather index, 20 to 33 (5 to 60 for individual fires). Fine fuel moisture code decreased, DMC had a mid-season peak, and DC increased through the fire season. Mean head fire intensities ranged from 10 to 28 MW m−1 in the boreal spruce fuel type, showing that most large fires exhibit crown fire behaviour. Intensities of individual fires can exceed 60 MW m−1. Most FWI System parameters did not show trends over the 41-year period because of large inter-annual variability. A changing climate is expected to create future weather conditions more conducive to fire throughout much of Canada but clear changes have not yet occurred.
The topological relationship between the large-scale attributes and local interaction patterns of complex networks
Authors: Vázquez A. , Dobrin R. , Sergi D. , Eckmann J.-P. , Oltvai Z. N. , Barabási A.-L.
Published: December 2004

Recent evidence indicates that the abundance of recurring elementary interaction patterns in complex networks, often called subgraphs or motifs, carry significant information about their function and overall organization. Yet, the underlying reasons for the variable quantity of different subgraph types, their propensity to form clusters, and their relationship with the networks' global organization remain poorly understood. Here we show that a network's large-scale topological organization and its local subgraph structure mutually define and predict each other, as confirmed by direct measurements in five well studied cellular networks. We also demonstrate the inherent existence of two distinct classes of subgraphs, and show that, in contrast to the low-density type II subgraphs, the highly abundant type I subgraphs cannot exist in isolation but must naturally aggregate into subgraph clusters. The identified topological framework may have important implications for our understanding of the origin and function of subgraphs in all complex networks.
Dynamics of cellular level function and regulation derived from murine expression array data
Authors: de Bivort Benjamin , Huang Sui , Bar-Yam Yaneer
Published: December 2004

A major open question of systems biology is how genetic and molecular components interact to create phenotypes at the cellular level. Although much recent effort has been dedicated to inferring effective regulatory influences within small networks of genes, the power of microarray bioinformatics has yet to be used to determine functional influences at the cellular level. In all cases of data-driven parameter estimation, the number of model parameters estimable from a set of data is strictly limited by the size of that set. Rather than infer parameters describing the detailed interactions of just a few genes, we chose a larger-scale investigation so that the cumulative effects of all gene interactions could be analyzed to identify the dynamics of cellular-level function. By aggregating genes into large groups with related behaviors (megamodules), we were able to determine the effective aggregate regulatory influences among 12 major gene groups in murine B lymphocytes over a variety of time steps. Intriguing observations about the behavior of cells at this high level of abstraction include: (i) a medium-term critical global transcriptional dependence on ATP-generating genes in the mitochondria, (ii) a longer-term dependence on glycolytic genes, (iii) the dual role of chromatin-reorganizing genes in transcriptional activation and repression, (iv) homeostasis-favoring influences, (v) the indication that, as a group, G protein-mediated signals are not concentration-dependent in their influence on target gene expression, and (vi) short-term-activating/long-term-repressing behavior of the cell-cycle system that reflects its oscillatory behavior.
Extensive phosphorylation of Smoothened in Hedgehog pathway activation
Authors: Zhang Chi , Williams Elizabeth H. , Guo Yurong , Lum Lawrence , Beachy Philip A.
Published: December 2004

The transmembrane protein Smoothened (Smo) is activated in response to the extracellular protein signal, Hedgehog (Hh), and transmits this state of pathway activity into the cell. Previous studies in
Drosophila
have correlated pathway activation with Smo accumulation and increased phosphorylation. Using immunopurification and mass spectrometry, we identify here 26 serine/threonine residues within the Smo C-terminal cytoplasmic tail that are phosphorylated in Hh-stimulated cells. By systematically substituting alanine or glutamic acid to block or simulate phosphorylation, we provide evidence for a functional role of collective phosphorylation of a subset of phosphoresidues in pathway activation. This role is indicated by the ability of altered Smo proteins to produce changes in transcription of Hh-responsive genes
in vivo
and in cultured cells. These altered Smo proteins also affect biochemical indicators of pathway activity, such as Smo accumulation and phosphorylation of other pathway components. The prevalence and arrangement of phosphoresidues within the Smo cytoplasmic tail at recognition sites for cAMP-dependent protein kinase and casein kinase 1 suggest a role for these kinases in Smo phosphorylation, and such a role is supported by the effects of manipulating kinase activities in cultured cells. Our studies confirm and extend previous studies showing a positive effect for cAMP-dependent protein kinase and uncover a positive role for casein kinase 1α in Hh pathway activation.
Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody
Authors: Colby David W. , Chu YiJia , Cassady John P. , Duennwald Martin , Zazulak Helen , Webster Jack M. , Messer Anne , Lindquist Susan , Ingram Vernon Martin , Wittrup K. Dane
Published: December 2004

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (V
L
)12.3, rescued toxicity in a neuronal model of HD. We also found that V
L
12.3 inhibited aggregation and toxicity in a
Saccharomyces cerevisiae
model of HD. V
L
12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets.
CrkII regulates focal adhesion kinase activation by making a complex with Crk-associated substrate, p130
Cas
Authors: Iwahara Toshinori , Akagi Tsuyoshi , Fujitsuka Yuki , Hanafusa Hidesaburo
Published: December 2004

CrkII is an adaptor protein possessing oncogenic potential despite the lack of an enzymatic domain. We investigated here the physiological functions of CrkII by studying its ability to induce anchorage-independent cell growth. We found that inhibition or null mutation of focal adhesion kinase (FAK) blocked the anchorage-independent growth induced by CrkII overexpression, indicating that FAK is a critical determinant of the transforming activity of CrkII. CrkII overexpression enhanced the autophosphorylation of FAK at Tyr-397 and tyrosine phosphorylation of p130
Cas
(Crk-associated substrate, Cas) upon stimulation of integrin by fibronectin. Moreover, the constitutive phosphorylation of FAK and Cas was observed in CrkII-overexpressing cells, even when they were in the suspended condition, consistent with the ability of CrkII to induce anchorage-independent growth. Using Cas-deficient cells, we showed Cas function to be essential for both the CrkII-induced phosphorylation of FAK (Tyr-397) and anchorage-independent cell growth. The CrkII-induced FAK autophosphorylation depended upon CrkII–Cas complex formation. Furthermore, we showed that CrkII knockdown resulted in defects in integrin-mediated events, such as cell spreading, haptotactic migration, and FAK autophosphorylation. The integrin-mediated FAK autophosphorylation was also reduced in Cas-deficient cells. These results suggest that the CrkII–Cas complex functions in integrin-mediated FAK activation signaling. Our findings show the importance of CrkII in integrin-mediated events, acting upstream of FAK to affect the activation of this kinase, which appears to have a central role in this pathway.
In vitro
characterization of IroB, a pathogen-associated
C
-glycosyltransferase
Authors: Fischbach Michael A. , Lin Hening , Liu David R. , Walsh Christopher T.
Published: December 2004

Pathogenic strains of
Escherichia coli
and
Salmonella enterica
modify the tricatecholic siderophore enterobactin (Ent) by glucosylation of three aryl carbon atoms, a process controlled by the
iroA
locus [Hantke, K., Nicholson, G., Rabsch, W. & Winkelmann, G. (2003)
Proc. Natl. Acad. Sci. USA
100, 3677–3682]. Here, we report the purification of the IroB protein and its characterization as the Ent
C
-glucosyltransferase. IroB transfers glucosyl groups from uridine-5′-diphosphoglucose to C5 of one, two, or three of the 2,3-dihydroxybenzoyl units of Ent to yield monoglucosyl-
C
-Ent (MGE), diglucosyl-
C
-Ent (DGE), and triglucosyl-
C
-Ent (TGE). DGE, also known as salmochelin S4, and macrolactone-opened derivatives have been isolated from the culture broths of
S. enterica
and uropathogenic
E. coli
[Bister, B., Bischoff, D., Nicholson, G. J., Valdebenito, M., Schneider, K., Winkelmann, G., Hantke, K. & Sussmuth, R. D. (2004)
Biometals
17, 471–481], but MGE and TGE have not been reported previously. IroB has a
k
cat
of ≈10 min
-1
for the first C-glucosylation and is distributive, with sequential conversion and buildup of MGE and then DGE. The C5 to C1′ regio-selectivity of the 2,3-dihydroxybenzoyl-glucose linkage at all three rings of TGE suggests a C5 carbanion,
para
to the C2 phenolate oxygen, as the carbon nucleophile in this novel enzymatic
C
-glucosylation.
In vitrofolate supplementation alleviates oxidative stress, mitochondria-associated death signalling and apoptosis induced by 7-ketocholesterol
Authors: Huang R.-F. S. , Yaong H.-C. , Chen S.-C. , Lu Y.-F.
Published: December 2004

Folate has recently been proposed as a new antioxidant. Folate supplementation may have a protective effect in counteracting oxidant-induced apoptotic damage. The present studies were undertaken to examine whether there is a direct link between folate levels, antioxidant capability and reduced apoptotic damage. Using anin vitrocellular model of 7-ketocholesterol (KC)-induced apoptosis, U937 cells were pre-cultured with a folate-deficient medium supplemented with various levels of folate (2–1500μmol/l) before treatment with 7-KC. Apoptotic markers, mitochondria-associated death signals and levels of reactive oxygen species were assayed. After treatment with 7-KC for 30h, low and high levels of folate supplementation significantly (P<0.05) reduced nuclear DNA loss. Only high levels of folate supplementation (>1000μmol/l) were effective in counteracting 7-KC-promoted apoptotic membrane phosphatidylserine exposure and DNA laddering. The attenuation of 7-KC-induced apoptotic damage by high-dose folate supplementation coincided with a partial normalization of mitochondria membrane potential dissipation, a suppression of cytochromecrelease and an inhibition of procaspase 3 activation. The prevention of mitochondrial dysfunctions and apoptotic processes was associated with antioxidant actions of high-dose folate by a marked scavenging of intracellular superoxide. Collectively, our present results demonstrate thatin vitrofolate supplementation exerts differentially protective effects against 7-KC-induced damage. High-dose supplementation alleviates oxidative stress, mitochondria-associated death signalling and apoptosis induced by 7-KC. However, thein vivorelevance is not clear and requires further study.
Vitamin K status of older individuals in northern China is superior to that of older individuals in the UK
Authors: Yan Liya , Zhou B. , Greenberg David , Wang Laura , Nigdikar Shailja , Prynne Celia , Prentice Ann
Published: December 2004

To explore whether differences in vitamin K nutrition might, at least in part, underlie differences in fracture incidence between Asian and European populations, the vitamin K status of older individuals in Shenyang, China (eighty-six men, ninety-two women) and in Cambridge, UK (sixty-seven men, sixty-seven women) was compared. Dietary information was collected by food questionnaire in Shenyang and food diary in Cambridge and used to estimate the intake and sources of phylloquinone. Fasting blood was analysed for phylloquinone, triacylglycerol, total osteocalcin (tOC) and undercarboxylated osteocalcin (expressed as percentage of tOC; %ucOC). The mean intakes of green leafy vegetables were 127 (SD 90) g/d in Shenyang and 39 (SD 48) g/d in Cambridge. The estimated phylloquinone intakes (geometric means) were 247 (95% CI 226, 270) μg/d in Shenyang and 103 (95% CI 94, 112) μg/d in Cambridge. Plasma phylloquinone concentrations (geometric means) were significantly higher in the Shenyang subjects (2·17 (95% CI 1·95, 2·42) nmol/l) than in the Cambridge subjects (0·69 (95% CI 0·63, 0·76) nmol/l; P<0·001). Plasma phylloquinone concentration was positively related to phylloquinone intake in both the Shenyang (coefficient 0·17 (SE 0·08); P=0·03) and Cambridge subjects (coefficient 0·29 (SE 0·10); P=0·005). tOC concentration and %ucOC (after adjusting for tOC) were significantly lower in the Shenyang than in the Cambridge subjects (tOC 25·2 (SE 4·2) % and %ucOC 68·5 (SE 10·0) % lower respectively; P<0·001). After adjusting for tOC and triacylglycerol, %ucOC was negatively related to plasma phylloquinone concentration in both the Shenyang (coefficient −0·41 (SE 0·11); P=0·0003) and Cambridge subjects (coefficient −0·17 (SE 0·07); P=0·02). The present study demonstrates that older individuals in northern China have a better vitamin K status compared with their British counterparts in Cambridge, UK.
Description and validation of the ActiReg®: a novel instrument to measure physical activity and energy expenditure
Authors: Hustvedt Bo-Egil , Christophersen Alf , Johnsen Lene R. , Tomten Heidi , McNeill Geraldine , Haggarty Paul , Løvø Arne
Published: December 2004

The ActiReg® (PreMed AS, Oslo, Norway) system is unique in using combined recordings of body position and motion alone or combined with heart rate (HR) to calculate energy expenditure (EE) and express physical activity (PA). The ActiReg® has two pairs of position and motion sensors connected by cables to a battery-operated storage unit fixed to a waist belt. Each pair of sensors was attached by medical tape to the chest and to the front of the right thigh respectively. The collected data were transferred to a personal computer and processed by a dedicated program ActiCalc®. Calculation models for EE with and without HR are presented. The models were based on literature values for the energy costs of different activities and therefore require no calibration experiments. The ActiReg® system was validated against doubly labelled water (DLW) and indirect calorimetry. The DLW validation demonstrated that neither EE calculated from ActiReg® data alone (EEAR) nor from combined ActiReg® and HR data (EEAR–HR) were statistically different from DLW results. The EEAR procedure causes some underestimation of EE >11 MJ corresponding to a PA level >2·0. This underestimation is reduced by the EEAR–HR procedure. The objective recording of the time spent in different body positions and at different levels of PA may be useful in studies of PA in different groups and in studies of whether recommendations for PA are being met. The comparative ease of data collection and calculation should make ActiReg® a useful instrument to measure habitual PA level and EE.
Independent of body adiposity, breast-feeding has a protective effect on glucose metabolism in young adult women
Authors: Diniz Juliana M. M. , Da Costa Teresa H. M.
Published: December 2004

The objective of the present study was to determine if any associations between reproductive experience and anthropometric or sub-clinical metabolic alterations of glucose metabolism exist. Sixty-seven women were recruited from the University of Brasilia Hospital and were evaluated at 12–18 months postpartum. Demographic, socio-economic, physical activity, anthropometric and health history (biochemical, reproductive) data were obtained. After a 12h overnight fast, a 2h oral glucose tolerance test was performed. Blood samples were collected at several points: at baseline, after intake of d-glucose solution (750g/l; 100ml) and every 30min thereafter. Blood glucose and lipids were measured by enzymic assays. Blood insulin was measured by RIA. In multiple regression analysis four dependent logarithmically transformed (logt) variables (increased area under the glucose curve (IAUGC), increased area under the insulin curve (IAUIC), insulin peak (IP), homeostasis model of assessment (HOMA)) were adjusted for parity, age, lactation index, BMI, percentage body fat (PBF), waist circumference, superior skinfold thickness sum:inferior skinfold thickness sum ratio and oral contraceptive use. PBF was positively associated with logt-IAUIC (P=0·004) and IP (P=0·006). However, the lactation index was negatively associated with logt-IAUIC (P=0·02) IAUGC and HOMA did not present significant associations. We conclude that during the postnatal period, independent of parity, body adiposity accumulation is associated with initial alterations in insulin secretion. Furthermore, independent of body adiposity, breast-feeding has a long-lasting protective effect on insulin response.
Dietary modulation of the human colonic microbiota: updating the concept of prebiotics
Authors: Gibson Glenn R. , Probert Hollie M. , Loo Jan Van , Rastall Robert A. , Roberfroid Marcel B.
Published: December 2004

Prebiotics are non-digestible (by the host) food ingredients that have a beneficial effect through their selective metabolism in the intestinal tract. Key to this is the specificity of microbial changes. The present paper reviews the concept in terms of three criteria: (a) resistance to gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption; (b) fermentation by intestinal microflora; (c) selective stimulation of the growth and/or activity of intestinal bacteria associated with health and wellbeing. The conclusion is that prebiotics that currently fulfil these three criteria are fructo-oligosaccharides, galacto-oligosaccharides and lactulose, although promise does exist with several other dietary carbohydrates. Given the range of food vehicles that may be fortified by prebiotics, their ability to confer positive microflora changes and the health aspects that may accrue, it is important that robust technologies to assay functionality are used. This would include a molecular-based approach to determine flora changes. The future use of prebiotics may allow species-level changes in the microbiota, an extrapolation into genera other than the bifidobacteria and lactobacilli, and allow preferential use in disease-prone areas of the body.
Reshaping the way we view vitamin D signalling and the role of vitamin D in health
Authors: Fleet James C. , Hong Jie , Zhang Zhentao
Published: December 2004

Although the biological requirement for vitamin D can be met by epidermal exposure to UV light, there are a number of conditions where this production does not occur or is not sufficient to meet biological needs. When this happens, vitamin D must be consumed and is a nutrient. However, two distinct observations have caused researchers to rethink certain dogma in vitamin D biology. First, it appears that in addition to the hormonally active form of 1,25 dihydroxyvitamin D (1,25(OH)2D), circulating levels of 25 hydroxyvitamin D have a critical importance for optimal human health. This and other data suggest that extra-renal production of 1,25(OH)2D contributes to Ca homeostasis and cancer prevention. Second, in addition to its role in the transcriptional activation of genes through the vitamin D receptor there is now compelling evidence that 1,25(OH)2D has a second molecular mode of action; the rapid activation of second-messenger and kinase pathways. The purpose of this second mode of action is only now being explored. The present review will discuss how these two areas are reshaping our understanding of vitamin D metabolism and action.