bluuebunny/crossref_metadata_2025 huggingface dataset:
56 PRODUCTION OF TRANSGENIC RECLONED MINIATURE PIGS EXPRESSING HUMAN DECAY ACCELERATING FACTOR
Authors: Lee S. H. , Jeong Y. W. , Jeon H. Y. , Kim S. , Kim J. H. , Koo O. J. , Lee E. G. , Park S. M. , Hashem M. D. A. , Hossein M. S. , Kang S. K. , Lee B. C. , Hwang W. S.
Published: December 2006

This study was performed to produce transgenic recloned miniature pigs expressing human decay-accelerating factor (hDAF) to overcome the hyperacute rejection of pig-to-human xenotransplantation. The expression vector, named hDAF-Neo, was constructed by subcloning the amplified 2.2 kb hDAF cDNA fragment into the NheI and NotI site of the pEGFP-N1 (Clontech, Palo Alto, CA, USA) vector containing the chicken beta-actin promoter and rabbit globin poly A without a EGFP fragment. Day 30 fetal fibroblast cells (GN0) were used for producing the newborn cloned pigs (GN1). GN1 fibroblasts cultured from the newborn cloned pig derived from GN0 were used for transfection of the hDAF-Neo plasmid using FuGENE-6� (Roche Diagnostics, Indianapolis, IN, USA). Transfected fibroblast cell colonies were selected with neomycin. All data were analyzed by one-way ANOVA and the protected least significant difference (LSD) test using general linear models in a statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Significant difference among the treatment groups was determined when the P value was less than 0.05. There was no significantly different development rate between GN0 and GN1 (12.8 vs. 13.0%) and between GN1-hDAF and GN2 (9.3 vs. 8.2%). Transfected recloned embryos (GN1-hDAF) had no significantly different blastocyst formation compared to normal (GN1). After embryo transfer, we obtained two transgenic recloned pigs. hDAF gene integration in transgenic recloned piglets was confirmed by polymerase chain reaction. Cultured fibroblasts of transgenic recloned pigs showed 1.0-2.3 times higher levels of hDAF protein than did human umbilical vein endothelial cells. The levels of C3 deposition on cultured fibroblast cells after incubation with 10% human serum were decreased in transgenic recloned pigs compared to normal miniature pigs. In conclusion, the recloning procedure can be used to produce multiple genetically modified cloned pigs without any severe epigenetic abnormality.
This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).
58 EFFICIENCY OF OVUM PICKUP AND EMBRYO PRODUCTION IN VITRO IN CLONED CATTLE
Authors: Lucas-Hahn A. , Lemme E. , Hadeler K.-G. , Sander H.-G. , Niemann H.
Published: December 2006

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning.

Table 1.
OPU and in vitro embryo production in cloned cattle
59 BOVINE OOCYTE RECONSTRUCTION FOLLOWING CRYOPRESERVATION AND GERMINAL VESICLE TRANSPLANTATION
Authors: Luciano A. M. , Franciosi F. , PerazzouI F. , Galbusera C. , Beretta M. S. , Lodde V. , Modina S.
Published: December 2006

The aim of the present study was to assess the maturational competence and the cytoskeletal pattern of bovine oocytes reconstructed by a germinal vesicle transplantation (GVT) technique using nuclear and/or cytoplasmic components from cryopreserved GV stage oocytes. Cumulus-oocyte complexes were isolated from slaughtered ovaries and denuded oocytes were cryopreserved by vitrification in open pulled straws (Vajta et al. 1998 Mol. Reprod. Dev. 51(1), 53-58) after cumulus cell removal (Modina et al. 2004 Eur. J. Histochem. 48(4), 337-346). Denuded oocytes were maintained in meiotic arrest with 0.5 mM of 3-isobutyl-1-methylxanthine, centrifuged for 20 min in for visualization of the germinal vesicle and then treated with 7.5 �g/mL of cytochalasin B for 20 min before enucleation. By means of micromanipulation and electrofusion procedures, female gametes were reconstructed from karyoplasts and cytoplasts obtained from either cryopreserved or freshly isolated oocytes, and then in vitro-matured for 24 h (Luciano et al. 2005 Mol. Reprod. Dev. 71(3), 389-397). After in vitro maturation (IVM), the nuclear stage was assessed by Hoechst 33342 staining and oocytes were fixed for analysis of the organization of microtubules and microfilaments, which provide the framework for chromosomal alignment and meiotic division. A total of 179 oocytes were reconstructed by the GVT procedure. All data were analyzed by chi-square test. No differences were observed in the reconstruction efficiency after fusion between fresh and cryopreserved oocytes (77.6% and 81.2%, respectively). The maturational competence of artificial gametes originated from cryopreserved oocytes was not different from that of gametes reconstructed with fresh oocytes (21.3% and 26.7%, respectively) and cytoskeleton organization analysis of artificial oocytes demonstrated that the correct distribution of microtubules and microfilaments was associated with a proper progression through metaphase II (MII) of meiotic division. However, the maturation rate of reconstructed oocytes was significantly lower than that of the control group, represented by untreated oocytes (86.7%; P < 0.05), and pre-enucleation procedures significantly affected the capability of oocytes to reach the MII stage of meiotic division (73.3%; P < 0.05). We conclude that both nuclear and cytoplasmic components derived from cryopreserved immature bovine oocytes are suitable for the GVT procedure, and that they generate oocytes able to progress to MII in vitro. The possibility of using cryopreserved immature oocytes as a source of nuclei and cytoplasm could help in applying the GV transfer procedure, both in research and clinical settings and in fertility preservation programs.
This work was supported by a PRIN 2003 MIUR Grant.
6 EXPRESSION PROFILING OF SINGLE BOVINE EMBRYOS REVEALS SIGNIFICANT EFFECTS OF IN VITRO MATURATION, FERTILIZATION AND CULTURE
Authors: Smith S. L. , Sung L.-Y. , Page R. , Henderson B. , Du F. , Everts R. E. , Nedambale T. , Rodriguez-Zas S. , Renard J.-P. , Lewin H. A. , Yang X. , Tian X. C.
Published: December 2006

Cattle and sheep embryos transferred after in vitro production are often afflicted by large offspring syndrome (LOS), which has been correlated with the presence of serum and/or cell co-culture. Previous research indicates that post-fertilization culture affects blastocyst quality and gene expression, and in vitro oocyte maturation and fertilization impact developmental competence. To dissect the effects of in vitro maturation, fertilization, and culture, we compared the expression profiles of single bovine blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF, n = 15); (2) in vivo maturation, in vivo fertilization, and in vitro culture (IVD, n = 14); and (3) in vivo maturation, fertilization, and development (AI, n = 14). For in vitro culture, the embryos were cultured for 2 days in CR1aa medium with bovine serum albumin (BSA) and then transferred to CR1aa with 10% fetal bovine serum (FBS) with cumulus cells until Day 7, at which time the embryos were vitrified. IVD zygotes were surgically collected from two superovulated Holstein donor cows 24 h post-insemination and cultured in the same system. To conduct expression profiling, total RNA was isolated from individual thawed embryos. The RNA was subjected to three rounds of amplification utilizing a previously adapted and validated T7 linear amplification protocol. Amplified RNA from each embryo and from a standard reference was indirectly labeled with Cy3 or Cy5 by dye swap and hybridized to a custom bovine cDNA microarray containing ~6300 unique genes. After Loess normalization, an ANOVA model (GeneSpring 6.1 and SAS 9.0) was used to identify differentially expressed genes. The P-values were adjusted for multiple comparisons using the false discovery rate approach, and a e2-fold differential criterion was applied. A subset of the differentially expressed genes was verified by real-time RT-PCR. The blastocyst rates for IVF and IVD embryos were 37% and 75%, respectively. There were 305, 365, and 200 genes differentially expressed between the AI and IVD, the IVF and IVD, and the AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and the IVD embryos, making these potential candidates for LOS. There were 61 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology categories 'RNA processing' and 'RNA binding' were over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on embryonic gene expression.
This work was supported by USDA grants to X.Y., H.A.L., and X.C.T.
68 GENE EXPRESSION COMPARISONS BETWEEN BOVINE IN VIVO AND CLONED EMBRYOS PRODUCED BY THREE DIFFERENT METHODS
Authors: Ruddock N. , Wilson K. , Cooney M. , Tecirlioglu R. , Hall V. , French A. , Holland M.
Published: December 2006

Developmental pathways in the mammalian embryo are profoundly influenced by the epigenetic interaction of the environment and the genome. Loss of epigenetic control has been implicated in aberrant gene expression and altered imprinting patterns with consequence to the physiology and viability of the conceptus. Bovine somatic cell nuclear transfer (SCNT) is contingent on in vitro culture, and both SCNT and culture conditions are known to induce changes in embryonic gene expression patterns. Using these experimental models, this study compared gene expression of Day 7 cloned blastocysts created from three different SCNT protocols using the same cell line, with Day 7 in vivo blastocysts to elucidate mechanisms responsible for variations in phenotypic outcomes. SCNT methods included: (1) traditional SCNT by subzonal injection (SI); (2) handmade cloning (HMC); and (3) modified serial nuclear transfer (SNT), developed within the group. Four imprinted genes (Grb10, Ndn, Nnat, and Ube3a), four chromatin remodeling genes (Cbx1, Cbx3, Smarca4, and Smarcb1) and two genes implicated in polycystic liver disease (Prkcsh and Sec63) were analyzed in single blastocysts from each treatment (n = 5). All blastocysts expressed Actin, Oct-4 and Ifn-tau. All genes were sequence verified. Several genes were expressed ubiquitously across all groups, including Ndn, Ube3a, Cbx1, Cbx3, and Smarcb1. Interestingly, Grb10 was not expressed in two HMCs and one SNT blastocyst. Nnat was weakly expressed in one in vivo blastocyst and in the majority of cloned blastocysts in all groups. Prkcsh and Sec63 were expressed in all but one HMC blastocyst. While gene expression patterns were mostly maintained following SCNT, the imprinted genes Nnat and Grb10 showed instances of differential or abnormal expression in SCNT embryos. The chromatin remodeling genes were maintained in all SCNT treatments. Prkcsh and Sec63 were both absent in one HMC blastocyst, with implications for liver dysfunction, a condition previously reported in abnormal cloned offspring. The variable mRNA expression following SCNT provides an insight into genetic and environmental factors controlling implantation, placentation, organ formation, and fetal growth.
7 PLASMA PROGESTERONE CONCENTRATIONS IN OVARIECTOMIZED COWS WITH INTRAVAGINAL DEVICES CONTAINING DIFFERENT LEVELS OF PROGESTERONE
Authors: Aviles M. , Cutaia L. , Videla Dorna I. , Aba M. , Bo G. A.
Published: December 2006

An experiment was designed to determine plasma progesterone (P4) concentrations in ovariectomized cows treated with either CIDR-B devices (Pfizer Animal Health, Hamilton, New Zealand) impregnated with 1.9 g of P4 or DIB devices (Syntex, Argentina) impregnated with 0.5 or 1.0 g of P4. Fifteen ovariectomized beef cows, weighing 350 to 450 kg and with a body condition score (BCS) 2.5 to 3.0 out of 5 were used. All cows were ovariectomized 30 days before the beginning of the trial and had plasma P4 concentrations <1 ng/mL (measured by radioimmunoassay (RIA)) 10 days before the trial. On Day 0, cows were stratified by weight and randomly assigned to one of three treatment groups to receive intravaginal devices containing 0.5 g of P4 (DIB 0.5), 1 g of P4 (DIB 1.0), or 1.9 g of P4 (CIDR-B). Blood samples were taken 12 and 24 h after intravaginal device insertion and then every 24 h until device removal on Day 7. Jugular blood samples were drawn into heparinized tubes and centrifuged within 20 min of collection; the plasma was harvested, frozen, and stored at −10°C. Progesterone was measured in plasma samples using a commercial solid-phase RIA (Coat-a-count; Diagnostic Products Corp., Los Angeles, CA, USA). All of the samples were analyzed at one time and the intra-assay coefficient of variation was 13%. Plasma P4 concentrations were compared among treatments by calculating the area under the curve over time and testing for differences by ANOVA. Plasma P4 profiles were also analyzed by ANOVA for repeated measures. Analysis of plasma P4 profiles revealed a day effect (P < 0.001) but not a treatment effect (P > 0.13) or a day by treatment interaction (P > 0.16; Table 1). The area under the curve did not differ (P = 0.95) among groups (DIB 0.5: 2236.4 ng, DIB 1.0: 2164.6 ng, and CIDR-B: 2266.8 ng). In all treatments, plasma P4 profiles were characterized by a sudden increase in P4 concentrations within 12 h of device insertion and a gradual decrease during the following several days. It was concluded that the use of CIDR-B devices impregnated with 1.9 g of P4 or DIB devices impregnated with 1.0 or 0.5 g of P4 did not result in different plasma P4 concentrations in ovariectomized cows. Further studies are needed to confirm these results in lactating and dry intact (not ovariectomized) cattle.

Table 1.
Mean plasma P4 concentrations (ng/mL ± SEM) in ovariectomized cows treated with intravaginal devices containing 0.5 g (DIB 0.5), 1.0 g (DIB 1.0), or 1.9 g (CIDR-B) of P4*
81 COMPARISON OF DEVELOPMENTAL ABILITY AMONG CLONED EMBRYOS WITH VARIOUS INDIVIDUAL RECIPIENT CYTOPLASMS IN BOVINE
Authors: Zhao J. G. , Yang X. Y. , Liu H. F. , Li H. , Huang S. Z. , Zeng Y. T.
Published: December 2006

Faithful reprogramming ensures the proper activation of genes during embryonic development of the somatic cell nuclear transfer (NT) in bovine. It is unambiguous that all these remodeling factors are presented in the oocyte cytoplasm (Du et al. 2002 Mol. Reprod. Dev. 63, 183–191). It will be interesting to determine if the recipient cytoplasms derived from individuals have different development ability and reprogramming competence during NT. Oocytes recovered by Ovum pickup from five Holstein heifers at 14 months of age were used as recipient cytoplasms. Cultured granulosa cells of the same origin were used as donor cells. Oocytes were enucleated at 20 h post-maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the reconstructed embryos were cultured in B2 medium (Laboratoire CCD, Paris, France) on a monolayer of Vero cells for 7 days. The oocyte number, development ability, and NT efficiency of recipient cytoplasm derived from each individual were compared (Table 1). Differences among individuals were verified using a chi-square test, SAS 6.12 version (SAS Institute, Cary, NC, USA). There were significant differences of survival after fusion and the rate of development to the blastocyst stage for embryos reconstructed with recipient cytoplasm from five different individual heifers (P < 0.05). However, maturation rate, fusion rate and cleavage rate of embryos reconstructed with recipient cytoplasm from five different individual heifers presented no significant differences (P > 0.05). Reconstructed embryos with recipient cytoplasm from one heifer (03025) showed a lower survival after fusion (61% vs. 80%, 86%, 77%, 91%) but a higher ability to develop to blastocyst stage (61% vs. 24%, 31%, 52%, 31%) than the embryos from the other four heifers. The current study showed that recipient cytoplasm from various individuals may present great differences in developmental ability in nuclear transfer. This may result from different compatibility between nucleus and mitochondria or the content of maternal RNA as well as proteins in the oocyte. Further studies are needed to elucidate the genetic factors that affect the reprogramming in nuclear transfer.

Table 1.
Nuclear transfer efficiency with various individual recipient cytoplasms
92 EVALUATION OF BOAR SPERM FUNCTIONALITY AFTER A CUSHIONED CENTRIFUGATION TECHNIQUE
Authors: Gadea J. , Martínez-Miró S. , Decuadro-Hansen G. , Matás C.
Published: December 2006

Separation of sperm from seminal plasma is required in most semen freezing procedures. Semen is typically subjected to centrifugation to concentrate sperm into a pellet and allow removal of the seminal plasma prior to dilution in freezing extender. Centrifugation is a relatively effective method to recover sperm, however, the process also causes considerable sperm damage. The use of a dense, inert, and isotonic solution as a cushion in the bottom of the centrifuge tube allows a greater centrifugation speed to be applied and results in greater sperm recovery. The aim of the present work was to evaluate the effects of this cushioned centrifugation technique on in vitro sperm viability and functionality. Sperm-rich fractions from 16 fertile boars were diluted and cooled to 15�C; then subsamples were centrifuged by one of two different techniques. A standard method (SM), 800 g for 10 min in 50-mL tubes (Westendorf et al. 1975 Dtsch. Tier�rztl. Wschr. 82, 261-267) and a cushioned method (CM), 1000 g for 20 min using 45 mL of diluted semen on 5 mL of an isotonic iodixanol solution (60% w/v gradient) were performed. Sperm samples were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378-391) to detect changes in lipid packing disorder of the plasma membrane. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of reactive oxygen species (ROS). A final set of sperm samples was stained with peanut aggultinin-fluorscein isothiocyanate (PNA-FITC) and propidium iodide to evaluate the acrosome reaction. All of these parameters were evaluated by flow cytometry before and after centrifugation. ANOVA analysis revealed that centrifugation altered lipid packing disorder and viability. Raw semen (RS) had a larger number of viable low lipid disorder sperm than centrifuged semen (RS = 86.9a vs. SM = 81.64b vs. CM = 80.6b, P < 0.01) and a decreased number of dead sperm cells (RS = 9.5a vs. SM = 15.0b vs. CM = 16.3b, P < 0.01). However, the cushioned and standard centrifugation methods yielded similar results for all the parameters measured. No significant differences were found for generation of ROS or in the number of sperm exhibiting the acrosome reaction. In conclusion, compared to the standard centrifugation method, this simple cushioned modification is a more efficient means of processing boar semen for freezing because significantly less sample losses are detected; also, it provides similar levels of sperm viability and functionality, and consequently a higher number of doses per ejaculation can be produced.
98 SUCCESSFUL PREGNANCIES FOLLOWING TRANSFER OF VITRIFIED PORCINE EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES
Authors: Hiruma K. , Ueda H. , Saito H. , Tanaka C. , Maeda N. , Kurome M. , Tomii R. , Ueno S. , Nagashima H.
Published: December 2006

To date only in vivo-produced embryos have successfully produced live piglets after cryopreservation. In this study, we aimed to produce piglets from vitrified embryos derived from in vitro matured (IVM) oocytes. Cumulus-oocyte complexes collected from ovaries obtained at a local slaughterhouse were matured for 44 to 45 h in NCSU23 MEDIUM supplemented with 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 75 �g/mL potassium penicillin G, 50 �g/mL streptomycin sulfate, and 10 IU/mL eCG/ hCG. These IVM oocytes were either activated for parthenogenesis or in vitro-fertilized (IVF). For IVF, oocytes were incubated with 5 � 106/mL of cryopreserved epididymal sperm in PGM-tac medium (Yoshioka et al. 2003 Biol. Reprod. 69, 2092-2099) for 20 h. Embryos were treated for removal of cytoplasmic lipid droplets (delipation; Nagashima et al. 1995 Nature 374, 416) at the 4- to 8-cell stages, around 50 to 54 h after activation or insemination. After culture in NCSU23 for 15 h, they were vitrified by the minimum volume cooling (MVC) method. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethylsulfoxide (DMSO), and 20% (v/v) calf serum for 4 min, followed by exposure to vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose, and 20% calf serum. Embryos were then loaded onto a Cryotop (Kitazato Supply Co., Tokyo, Japan) and immediately plunged into liquid nitrogen. Vitrified embryos were examined for viability in vitro and in vivo after warming. Their in vitro developmental competence was compared to that of corresponding control (nonvitrified) embryos. Vitrified 4- to 8-cell stage embryos, both parthenogenetic and IVF, showed developmental competence into blastocysts comparable to that of control embryos (parthenogenetic: 46.8%, 36/77 vs. 51.7%, 31/60; IVF: 40.0%, 30/75 vs. 44.3%, 35/79). Of four surrogate gilts that received a total of 251 vitrified parthenogenetic embryos, three became pregnant and had 20 fetuses (8.0%, 22 to 23 days old). Three surrogates gilts that received 267 vitrified IVF embryos all became pregnant. Of those, the one that received 47 embryos was confirmed to have eight fetuses (17.0%, 22 days old) by autopsy. The other two were examined by ultrasonography at 56 and 95 days of gestation and found to be pregnant. These results suggest that porcine embryos derived from IVM oocytes have a potential to develop into live offspring after delipation and MVC vitrification.
This study was supported by PROBRAIN.
Contrasts between Antarctic and Arctic ozone depletion
Authors: Solomon Susan , Portmann Robert W. , Thompson David W. J.
Published: January 2007

This work surveys the depth and character of ozone depletion in the Antarctic and Arctic using available long balloon-borne and ground-based records that cover multiple decades from ground-based sites. Such data reveal changes in the range of ozone values including the extremes observed as polar air passes over the stations. Antarctic ozone observations reveal widespread and massive local depletion in the heart of the ozone “hole” region near 18 km, frequently exceeding 90%. Although some ozone losses are apparent in the Arctic during particular years, the depth of the ozone losses in the Arctic are considerably smaller, and their occurrence is far less frequent. Many Antarctic total integrated column ozone observations in spring since approximately the 1980s show values considerably below those ever observed in earlier decades. For the Arctic, there is evidence of some spring season depletion of total ozone at particular stations, but the changes are much less pronounced compared with the range of past data. Thus, the observations demonstrate that the widespread and deep ozone depletion that characterizes the Antarctic ozone hole is a unique feature on the planet.
Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases
Authors: Cho HyunDae D. , Verlinde Christophe L. M. J. , Weiner Alan M.
Published: December 2006

CCA-adding enzymes build and repair the 3′-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson–Crick edges of incoming CTP and ATP. Guided by the class II
Bacillus stearothermophilus
CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related
Aquifex aeolicus
CC- and A-adding enzymes into UU- and G-adding enzymes and
Escherichia coli
poly(A) polymerase into a poly(G) polymerase; and we transformed the
B. stearothermophilus
CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the
B. stearothermophilus
CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2′ hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 Å away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3′ end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.
Promoting motions in enzyme catalysis probed by pressure studies of kinetic isotope effects
Authors: Hay Sam , Sutcliffe Michael J. , Scrutton Nigel S.
Published: January 2007

Use of the pressure dependence of kinetic isotope effects, coupled with a study of their temperature dependence, as a probe for promoting motions in enzymatic hydrogen-tunneling reactions is reported. Employing morphinone reductase as our model system and by using stopped-flow methods, we measured the hydride transfer rate (a tunneling reaction) as a function of hydrostatic pressure and temperature. Increasing the pressure from 1 bar (1 bar = 100 kPa) to 2 kbar accelerates the hydride transfer reaction when both protium (from 50 to 161 s
−1
at 25°C) and deuterium (12 to 31 s
−1
at 25°C) are transferred. We found that the observed primary kinetic isotope effect increases with pressure (from 4.0 to 5.2 at 25°C), an observation incompatible with the Bell correction model for hydrogen tunneling but consistent with a full tunneling model. By numerical modeling, we show that both the pressure and temperature dependencies of the reaction rates are consistent with the framework of the environmentally coupled tunneling model of Kuznetsov and Ulstrup [Kuznetsov AM, Ulstrup J (1999)
Can J Chem
77:1085–1096], providing additional support for the role of a promoting motion in the hydride tunneling reaction in morphinone reductase. Our study demonstrates the utility of “barrier engineering” by using hydrostatic pressure as a probe for tunneling regimes in enzyme systems and provides added and independent support for the requirement of promoting motions in such tunneling reactions.
The IL-15/IL-15Rα on cell surfaces enables sustained IL-15 activity and contributes to the long survival of CD8 memory T cells
Authors: Sato Noriko , Patel Hiral J. , Waldmann Thomas A. , Tagaya Yutaka
Published: January 2007

We previously described unique features of the IL-15 receptor (IL-15R)α. IL-15Rα by itself forms stable complexes with IL-15 on cell surfaces and presents IL-15in transto neighboring natural killer/T cells. Moreover, the membrane IL-15/IL-15Rα complexes (membIL-15) undergo endosomal internalization but survive lysosomal degradation, allowing the complexes to recycle back to the cell surface. Here, we show that membIL-15+cells act as a persistent source of IL-15 for the surrounding microenvironment (intercellular reservoir effect). Additionally, membIL-15+cells give rise to augmented retention of IL-15 in the circulation as well as in tissues. Curiously, IL-15 retention was particularly associated with lungs, rather than with lymph nodes, in normal unstimulated mice, which correlated with the preferential homing of antigen-specific CD8 T cells to lungs during their contraction phase in an IL-15Rα-dependent manner. Furthermore, membIL-15, unlike soluble IL-15, caused sustained IL-15 signal transduction in the target cells. Collectively, these characteristics define IL-15 as a unique cytokine with prolongedin vivosurvival and sustained biological action on the target cells, which may account for the proposed persistent action of IL-15 that helps the long-term survival of functional CD8 memory T cellsin vivo.
Cochlear Cytokine Gene Expression in Murine Acute Otitis Media
Authors: Ghaheri Bobak A. , Kempton J Beth , Pillers De‐Ann M. , Trune Dennis R.
Published: January 2007

Objective: Recurrent acute otitis media (AOM) causes sensorineural hearing loss by unknown mechanisms. It is widely accepted that inflammatory cytokines diffuse across the round window membrane to exert cytotoxic effects. This study addresses whether inner ear cells are capable of expressing genes for inflammatory cytokines.

Study Design: The authors conducted a prospective animal study.

Methods: BALB/C mice underwent transtympanic injection of heat‐killed Haemophilus influenzae to create an acute inflammatory response. These mice were compared with a control group in addition to a group of uninjected mice found to have otomicroscopic changes consistent with persistent or chronic otitis media. The cochleas of these mice were obtained, their RNA harvested, and cytokine gene expression analyzed using prefabricated cDNA arrays.

Results: Four groups of mice (control, 3‐day postinjection, 7‐day postinjection, and mice with chronic otitis media) with five mice in each group were analyzed. Numerous classes of genes were found to be upregulated or downregulated by more than twofold. Some genes differed from control mice by more than 10‐fold. These genes included numerous fibroblast growth factors, interleukins, tumor necrosis factors, and colony‐stimulating factors.

Conclusion: The genes of numerous inflammatory cytokines are either up‐ or downregulated by murine inner ear cells in response to either acute or chronic inflammation of the middle ear. This study provides a novel site of production of cytokines that may be responsible for the damage seen in sensorineural hearing loss.
A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana
Authors: Rajagopalan Ramya , Vaucheret Hervé , Trejo Jerry , Bartel David P.
Published: December 2006

To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.
Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway
Authors: Shiojima Ichiro , Walsh Kenneth
Published: December 2006

Postnatal growth of the heart is primarily achieved through hypertrophy of individual myocytes. Cardiac growth observed in athletes represents adaptive or physiological hypertrophy, whereas cardiac growth observed in patients with hypertension or valvular heart diseases is called maladaptive or pathological hypertrophy. These two types of hypertrophy are morphologically, functionally, and molecularly distinct from each other. The serine/threonine protein kinase Akt is activated by various extracellular stimuli in a phosphatidylinositol-3 kinase-dependent manner and regulates multiple aspects of cellular functions including survival, growth and metabolism. In this review we will discuss the role of the Akt signaling pathway in the heart, focusing on the regulation of cardiac growth, contractile function, and coronary angiogenesis. How this signaling pathway contributes to the development of physiological/pathological hypertrophy and heart failure will also be discussed.
The establishment of neuronal properties is controlled by Sox4 and Sox11
Authors: Bergsland Maria , Werme Martin , Malewicz Michal , Perlmann Thomas , Muhr Jonas
Published: December 2006

The progression of neurogenesis relies on proneural basic helix–loop–helix (bHLH) transcription factors. These factors operate in undifferentiated neural stem cells and induce cell cycle exit and the initiation of a neurogenic program. However, the transient expression of proneural bHLH proteins in neural progenitors indicates that expression of neuronal traits must rely on previously unexplored mechanisms operating downstream from proneural bHLH proteins. Here we show that the HMG-box transcription factors Sox4 and Sox11 are of critical importance, downstream from proneural bHLH proteins, for the establishment of pan-neuronal protein expression. Examination of a neuronal gene promoter reveals that Sox4 and Sox11 exert their functions as transcriptional activators. Interestingly, the capacity of Sox4 and Sox11 to induce the expression of neuronal traits is independent of mechanisms regulating the exit of neural progenitors from the cell cycle. The transcriptional repressor protein REST/NRSF has been demonstrated to block neuronal gene expression in undifferentiated neural cells. We now show that REST/NRSF restricts the expression of Sox4 and Sox11, explaining how REST/NRSF can prevent precocious expression of neuronal proteins. Together, these findings demonstrate a central regulatory role of Sox4 and Sox11 during neuronal maturation and mechanistically separate cell cycle withdrawal from the establishment of neuronal properties.
Mitf regulation of Dia1 controls melanoma proliferation and invasiveness
Authors: Carreira Suzanne , Goodall Jane , Denat Laurence , Rodriguez Mercedes , Nuciforo Paolo , Hoek Keith S. , Testori Alessandro , Larue Lionel , Goding Colin R.
Published: December 2006

It is widely held that cells with metastatic properties such as invasiveness and expression of matrix metalloproteinases arise through the stepwise accumulation of genetic lesions arising from genetic instability and “clonal evolution.” By contrast, we show here that in melanomas invasiveness can be regulated epigenetically by the microphthalmia-associated transcription factor, Mitf, via regulation of the DIAPH1 gene encoding the diaphanous-related formin Dia1 that promotes actin polymerization and coordinates the actin cytoskeleton and microtubule networks at the cell periphery. Low Mitf levels lead to down-regulation of Dia1, reorganization of the actin cytoskeleton, and increased ROCK-dependent invasiveness, whereas increased Mitf expression leads to decreased invasiveness. Significantly the regulation of Dia1 by Mitf also controls p27Kip1-degradation such that reduced Mitf levels lead to a p27Kip1-dependent G1 arrest. Thus Mitf, via regulation of Dia1, can both inhibit invasiveness and promote proliferation. The results imply variations in the repertoire of environmental cues that determine Mitf activity will dictate the differentiation, proliferative, and invasive/migratory potential of melanoma cells through a dynamic epigenetic mechanism.
London audit of drug‐related overdose deaths: characteristics and typology, and implications for prevention and monitoring
Authors: Hickman Matthew , Carrivick Sandra , Paterson Susan , Hunt Neil , Zador Deborah , Cusick Linda , Henry John
Published: December 2006

Objectives  To describe the circumstances and draft a typology of drug‐related overdose deaths.

Setting  London, 2003.

Methods  An audit of 148 drug overdose deaths (involving heroin, methadone, dihydrocodeine, cocaine, amphetamine or MDMA) investigated by coroners during 2003. Information extracted on toxicology, pathology and circumstances were used to identify drug(s) implicated in the death.

Results  Poly‐ or multiple drug use was detected in the majority of deaths with at least 69 different combinations, including 66% for heroin and 42% for cocaine. Six categories of death were identified involving an opiate (100, 68%); cocaine (14, 9%); other controlled drug (five, 3%); mixed drug overdose (18, 12%); other prescribed drug (five, 3%); and other causes (seven, 5%). A witness was present and the death was not instantaneous in 92 (61%) cases, although evidence in the coronial file suggested that in the majority of cases the overdose went unnoticed until too late to intervene. In all, 15 (one in 10) of the deceased were released from prison within 3 months of death; and 37 (one in four) were reported as in receipt of a methadone prescription.

Conclusions  Perhaps for the first time in the United Kingdom cocaine was detected in more drug overdose deaths than methadone. However, reducing heroin use is central to the prevention of drug‐related deaths. We recommend that overdose prevention encompasses strategies to encourage a ‘mutual duty of care’ among problem drug users, and in the United Kingdom further investigation of the relationship of methadone treatment failures on overall trends in drug‐related deaths is merited.
Association of serum fetuin‐A with carotid arterial stiffness
Authors: Mori Katsuhito , Emoto Masanori , Araki Takahiro , Yokoyama Hisayo , Teramura Megumi , Lee Eiko , Motoyama Koka , Koyama Hidenori , Shoji Tetsuo , Inaba Masaaki , Nishizawa Yoshiki
Published: December 2006

Objective   Fetuin‐A is a circulating glycoprotein which is well characterized as an inhibitor of ectopic calcification. Vascular calcification commonly found in chronic kidney disease (CKD) patients is a predictor of cardiovascular death. Recently, several groups have demonstrated that low fetuin‐A levels are associated with mortality in uraemic patients, possibly through regulation of vascular calcification. However, the physiological significance of fetuin‐A in atherosclerosis remains unknown, except in specific conditions, such as vascular calcification in CKD patients. The objective of this study was to investigate the association between serum fetuin‐A levels and arterial stiffness, a functional property of atherosclerosis, in healthy subjects.

Patients and measurements   The study subjects comprised 141 healthy subjects. We measured serum fetuin‐A levels and stiffness parameter β for the common carotid artery, which was assessed by ultrasound using a phase‐locked echo‐tracking system.

Results   Simple regression analyses indicated that serum fetuin‐A levels were significantly correlated with stiffness parameter β (r = 0·200, P = 0·018). Multiple regression analyses showed that, besides age, fetuin‐A (β = 0·166, P = 0·033) independently contribute to the stiffness parameter β (R2 = 0·310, P < 0·0001).

Conclusions   Serum fetuin‐A level is associated with carotid arterial stiffness, independent of known atherogenic factors in healthy subjects.
Congenital adrenal hyperplasia and P450 oxidoreductase deficiency
Authors: Krone Nils , Dhir Vivek , Ivison Hannah E. , Arlt Wiebke
Published: December 2006

Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders, which are usually due to inactivating mutations in single enzymes involved in adrenal steroid biosynthesis. The characteristics of the biochemical and clinical phenotype depend on the specific enzymatic defect. In 21‐hydroxylase and 11β‐hydroxylase deficiency only adrenal steroidogenesis is affected, whereas a defect in 3β‐hydroxysteroid dehydrogenase or 17α‐hydroxylase also involves gonadal steroid biosynthesis. Recently, mutations in the electron donor enzyme P450 oxidoreductase were identified as the cause of CAH with apparent combined 17α‐hydroxylase and 21‐hydroxylase deficiency, thereby illustrating the impact of redox regulation enzymes on steroidogenesis. P450 oxidoreductase deficiency (ORD) has a complex phenotype including two unique features not observed in any other CAH variant, skeletal malformations and severe genital ambiguity in both sexes. Despite invariably low circulating androgens, females with ORD may present with virilized genitalia and mothers may suffer from virilization during pregnancy. This apparently contradictory finding may be explained by the existence of an alternative pathway in human androgen biosynthesis, with important implications for physiology and pathophysiology. This review discusses the biochemical and clinical presentation and the genetic and functional basis of the currently known CAH variants, with a specific focus on ORD.
Longitudinal analysis of inpatient care utilization among people with intellectual disabilities: 1999–2002
Authors: Loh C.‐H. , Lin J.‐D. , Choi I.‐C. , Yen C.‐F. , Hsu S.‐W. , Wu J.‐L. , Tang C. C.
Published: December 2006

Background  There has been no longitudinal study in Taiwan to identify the nature and the scale of medical care utilization of people with intellectual disabilities (IDs) up to the present. The aim of this study is to describe inpatient utilization among people under ID care in institutions in order to identify the pattern of medical care needs and the factors affecting utilization in Taiwan.

Method  The subject cohort was 168 individuals with ID who were cared for by a large public disability institution from 1999 to 2002 in Taipei, Taiwan.

Results  On the examination of the inpatient care that these persons underwent, it was found that these individuals had a heightened need (inpatient rate: 10.1–14.9%) for inpatient care compared with the general population with disabilities (9.37%) in Taiwan. The main reasons for hospitalization were pneumonia, gastrointestinal disorders, cellulites, orthopaedic problems, epilepsy and bronchitis. Using the full model of Generalized Estimating Equations for inpatient care utilization, the factors including low income family, living in an institution, being a subject with cerebral palsy and being a high outpatient user all influenced the use of inpatient care.

Conclusions  This study highlights that health authorities need to promote health planning more in order to ensure an excellent quality of health monitoring and health promotion among people with ID cared for by institutions.
A tale of two matrices: multivariate approaches in evolutionary biology
Authors: BLOWS M. W.
Published: December 2006

Two symmetric matrices underlie our understanding of microevolutionary change. The first is the matrix of nonlinear selection gradients (γ) which describes the individual fitness surface. The second is the genetic variance–covariance matrix (G) that influences the multivariate response to selection. A common approach to the empirical analysis of these matrices is the element‐by‐element testing of significance, and subsequent biological interpretation of pattern based on these univariate and bivariate parameters. Here, I show why this approach is likely to misrepresent the genetic basis of quantitative traits, and the selection acting on them in many cases. Diagonalization of square matrices is a fundamental aspect of many of the multivariate statistical techniques used by biologists. Applying this, and other related approaches, to the analysis of the structure ofγandGmatrices, gives greater insight into the form and strength of nonlinear selection, and the availability of genetic variance for multiple traits.
Mitochondrial Activity in Orobanche cernua from Different Regions of the Scape and as Influenced by Host
Authors: Singh P.
Published: December 2006

Orobanche cernua, a holoparasite, was harvested from different hosts, namely, Solanum melongena, Petunia hybrida, Lycopersicum esculentum, Solanum nigrum and Datura metel. Mitochondrial particles were isolated and they were evaluated in terms of the marker enzyme, cytochrome c oxidase, and protein in the mitochondrial fraction. Protein levels in whole homogenate and mitochondrial fraction of parasite growing on different host plants did not vary significantly, whereas the recovery of protein (% of whole homogenate) in the mitochondrial fraction of parasite growing on D. metel was higher. Cytochrome c oxidase activity in parasites growing on the three host plants varied, being highest when the parasite grew on S. melongena, followed sequentially by that on P. hybrida and D. metel. Protein in whole homogenate and mitochondrial fraction was significantly lower, 25–36% and 15–33%, respectively, in distal region when compared with the proximal region. Similarly, cytochrome oxidase and respiratory activity was significantly lower, 23–34% and 18–23%, respectively, in the distal region of the parasite. In addition, variations in cytochrome oxidase and respiratory activity in the proximal and distal regions of the parasite growing on different hosts was also significant. Results indicated that mitochondria in haustoria‐bearing proximal region of Orobanche scape play a special role to meet the metabolic demand of the parasite.