bluuebunny/crossref_metadata_2025 huggingface dataset:
76 SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION IN EXTENDERS WITH OR WITHOUT EGG YOLK
Authors: Saenz J. R. , Dumas C. , Dresser B. L. , Gómez M. C. , Godke R. A. , Pope C. E.
Published: December 2008

Our purpose was to compare in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST egg-yolk buffered extender (TYB) with that obtained by use of clear Tris-citrate and HEPES-buffered extenders containing BSA. Testes were transported to the lab in HEPES saline; epididymides were dissected in HEPES-199 medium (HE-199) and repeatedly sliced. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolater, Irving Scientific, Santa Ana, CA), and centrifuged at 600g for 20 min. Aliquots of the sperm pellet were extended in TYB, Human Sperm Preservation Medium (HSPM), or Tris-citrate + 10% BSA (TCBSA). After cooling to 4°C, samples were diluted 1:1 with extender + 12% glycerol in 4 steps as modified from Gao DY et al. 1995 Hum. Reprod. 10, 1109–1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of HE-199 in 7 steps as modified from Gao DY et al. 1995 Hum. Reprod. 10, 1109–1122, centrifuged at 200g for 10 min and pellets resuspended in HE-199. Motility (MOT, phase contrast, 37°C), membrane integrity (MI, SYBR 14–PI), and acrosomal status (AS, FITC–PNA) were evaluated at 0 h, after gradual cooling to 4°C, and after freezing at 0 h and 3 h post-thaw (37°C). Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in either TYB or HSPM in droplets (1 million sperm mL–1) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured using a 3-step system (Pope CE et al. 2006 Theriogenology 66, 59–71) until blastocyst development was evaluated (Day 8). There were no treatment differences at any time/temperature point for the 3 sperm parameters evaluated (one-way ANOVA; P > 0.05). As shown in Table 1, sperm motility in TCBSA and HSPM decreased by 20% after cooling to 4°C and another 20% after freezing, whereas motility in TYB was maintained after cooling and decreased <30% after freezing. Membrane integrity and acrosomal status values were 12 to 15% greater at collection, at 4°C and at 0 h post-thaw, and 25% greater at 3 h post-thaw than were the motility values. Cleavage frequency and blastocyst development rate of 203 IVM oocytes after IVF using sperm frozen in TYB and HSPM was 36 v. 33% and 50 v. 44%, respectively. In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in cryoprotectant solutions that do not contain egg yolk.

Table 1.Motility, membrane integrity and acrosomal status of cat epididymal sperm after cryo-storage
8 EFFECT OF CIRCULATING LEPTIN LEVELS AT SYNCHRONIZATION ON SUBSEQUENT FIXED-TIMED ARTIFICIAL INSEMINATION PREGNANCY RATES IN CROSSBRED BEEF HEIFERS
Authors: Gentry G. T. , Pitchford J. A. , Chiasson M. , Gentry L. R. , Bondioli K. R. , Thompson D. L. , Godke R. A.
Published: December 2008

It is generally accepted that reproductive efficiency is the limiting factor in the profitability of most livestock operations. The heritabilities of reproductive traits are typically low, so most advances in reproductive efficiency must be achieved through changes in management practices. Recent research indicates that leptin may play a role in the reproductive processes for many domesticated livestock species. Leptin has been shown to affect nutrient intake, metabolic efficiency, and animal adiposity, and may be associated with age at puberty onset and the length of the postpartum interval in cattle. Therefore, the objective of this study was to determine whether circulating leptin concentrations during estrous synchronization would affect subsequent pregnancy rates after fixed-timed AI (FTAI) in crossbred beef heifers. Before the initiation of the spring breeding season, 54 crossbred (Red Angus × Simmental) long yearling heifers, averaging 326 ± 6.3 kg with a mean body condition score (BCS) of 5.2 ± 0.33, a mean reproductive tract score of 2.1 ± 0.57, and a mean ovary score of 2.7 ± 0.76, were synchronized with a controlled internal drug-release (CIDR) vaginal implant and 2 mg of estradiol benzoate (EB) on Day 0, a 25-mg injection of prostaglandin and CIDR removal on Day 7, and 2 mg of EB on Day 8, with FTAI occurring 52 hours post-CIDR removal. Blood samples were collected via jugular venipuncture 4 times: twice during the synchronization protocol starting 2 days post-CIDR insertion, once at insemination, and once 4 days after insemination. All females were inseminated with frozen–thawed semen from the same fertile bull. Pregnancy was determined 45 days postinsemination via transrectal ultrasonography. Twenty females (37%) were confirmed pregnant to FTAI. There was no difference in average weights (324 ± 6.6 v. 323 ± 3.7 kg), BCS (5.1 ± 0.1 v. 5.2 ± 0.07), reproductive tract scores (2.2 ± 0.5 v. 2.1 ± 0.5) or ovary scores (2.7 ± 0.2 v. 2.7 ± 0.1) for the pregnant and nonpregnant females, respectively. However, those females that were pregnant from FTAI had higher (P = 0.02) mean plasma leptin concentrations (3.33 ± 0.22 ng mL–1) compared with heifers that did not conceive to FTAI (2.66 ± 0.19 ng mL–1) across all sampling days. In addition, as early as 2 days post-CIDR insertion, females that became pregnant from FTAI tended to have higher (P = 0.13) plasma leptin concentrations (2.76 ± 0.18 ng mL–1) compared with those heifers that did not conceive to FTAI (2.19 ± 0.15 ng mL–1). These results indicate that circulating plasma leptin levels in beef cattle during synchronization may be associated with subsequent fertility and might be a useful tool to select females to increase the efficacy of FTAI protocols in beef cattle.
89 MALE-TO-FEMALE SEX REVERSAL IN WOLF DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER WITH 78,XY KARYOTYPE
Authors: Kang J. T. , Oh H. J. , Hong S. G. , Park J. E. , Kim D. Y. , Jang G. , Kim M. K. , Lee B. C.
Published: December 2008

Normal sexual differentiation relies on a complex cascade of numerous genes, many of which have yet to be identified. Gene defects can cause disorders of sexual development with varying severity. Among them, SRY (sex-determining region on Y chromosome) genes are involved in the early steps of maleness determination. In human, mutations of the SRY gene have been diagnosed in up to 20% cases of females with XY karyotype. There have been a few instances of intersexuality such as true hermaphrodites or pseudo hermaphrodites, but no reports yet in canid of XY male-to-female sex reversal. Recently, we reported the cloning of endangered male gray wolves (Canis lupus) from somatic cells collected postmortem (Oh HJ et al. 2008 Theriogenology). In this study, the 3 wolves were delivered with normal karyotype (78,XY), but 1 wolf had female external genitalia such as a vulva and clitoris. For further diagnosis, we conducted macroscopic, radiologic, surgical, histological, and cytogenetic study. On gonadotropin-releasing hormone stimulation test, the blood serum testosterone concentration was low (<1 ng mL–1). Imaging diagnosis by radiography in the form of uterogram and by ultrasonography revealed a normal uterus and bilateral ovary-like structures located caudal to the kidney. During exploratory laparotomy, we found a normal uterus and bilateral ovary-like mass. Histopathological analysis showed the uterus and ovary to be normal, but no evidence of the presence of testicular tissue. The molecular analysis of the SRY genes with a direct DNA sequencing technique detected nucleotide changes more than one position in this affected animal compared with that of the control groups (cell donor and other female litter mates) although at a low rate. However, we did not yet analyze the nature of this mutation such as amino acid change, location within gene, protein function, and so on, so it will be needed further studies. These results indicate that this abnormal sex development of the studied wolf was probably caused by SRY gene mutations. Herein, we reported the case of a cloned wolf with normal ovaries and uterus having a 78,XY karyotype. Further studies are required to elucidate whether this mutation in the SRY gene could impair the normal function of the SRY protein.

This study was financially supported by the Korean MEST, through KOSEF (grant # M10625030005-08N250300510) and the BK21 program for Veterinary Science, and SNU foundation(Benefactor; RNL BIO)/Hanwha L&C.
96 AN IMMUNOHISTOCHEMICAL STUDY ON MARKERS OF PLURIPOTENCY AND DNA METHYLATION IN THE DEVELOPING PORCINE GERM LINE
Authors: Vejlsted M. , Hyldig S. M. W. , Maddox-Hyttel P.
Published: December 2008

Reprogramming of the germ line genome is a fundamental molecular process involving DNA demethylation. This has been demonstrated in the mouse (Seki Y et al. 2005 Dev. Biol. 278, 440–458), but has not yet been studied in the pig. From a large collection of porcine embryos/fetuses 2 to 7 weeks post-insemination (p.i.), a total of 35 randomly selected specimens from the end of the second (n = 10), third (n = 10), fourth (n = 5), and seventh (n = 10) gestational week were selected for an immunohistochemical study on pluripotency markers and DNA methylation in the developing germ line. Intact embryos and isolated developing gonads were paraffin embedded, sectioned (5 to 15 μm) and evaluated for the expression of markers of pluripotency [OCT4 (sc-8628, Santa Cruz Biotech., Santa Cruz, CA), Nanog (500-P236, PeproTech EC, Rocky Hill, NJ), and SOX2 (MAB2018, R&D Systems, Wiesbaden, Germany)], DNA methylation [5-methyl cytidine (ab10805, Abcam, Cambridge, MA)], and meiosis [SCP-3 (generous gift from C. Heyting)]. Heat-induced epitope retrieval (HIER) in an alkaline (pH 8.2) EDTA buffer (Shi SR et al. 2001 J. Histochem. Cytochem. 49, 931–937) and confocal laser scanning microscopy allowed for the evaluation of germ cells co-expressing OCT4 and 5-methyl cytidine. The expression of Nanog and SOX2 was found to be better visualized using HIER in an acidic (pH 6.0) citrate buffer. Isolated and clustered primordial germ cells (PGC) were identified by OCT4 labeling early during gastrulation in embryos around 2 weeks of age p.i. The amount of methylated DNA in PGC appeared similar to that in the nuclei of neighboring somatic cells at this stage. During colonization of the genital ridges, in embryos at the end of the third gestational week, this global DNA methylation status seemed to markedly decrease in PGC, remaining low in the gonadal maturing germ cells. Around onset of meiosis, in fetuses at the seventh gestational week, germ cells in 3 out of 5 female specimens studied had ceased to express markers of pluripotency. In contrast, such markers appeared to be retained in germ cells of male siblings. In conclusion, expression of pluripotency markers during porcine germ line development appears similar to what has been described in the mouse with expression ceasing at the beginning of meiosis in the female but not in the male fetus. Further, the timing of germ line DNA demethylation appears similar between the 2 species. In the mouse, PGC entering the genital ridges soon initiate meiosis, whereas in the pig, these events are separated by a 3-week period. The connection between porcine germ line pluripotency and DNA methylation status during the third to fourth week of development p.i. is presently being thoroughly investigated.
97 POSITION CHANGES IN THE RELATIONSHIP BETWEEN FIRST POLAR BODY AND CHROMOSOME OF GOLDEN HAMSTER OOCYTES
Authors: Wang L. Y. , Li D. X. , Li Z. Y.
Published: December 2008

The golden hamster represents an attractive species for studying reproductive physiology, oncology, genetics, and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, positional changes in the relationship between the first polar body (FPB) and chromosomes of golden hamster oocytes were examined under different conditions. 1) Female hamsters (6 weeks of age) superovulated with eCG (30 IU, i.p.) followed by hCG, (30 IU, i.p.) at intervals of 72 h were sacrificed at different times (13.5, 18, and 23 h) following hCG injection. The cumulus–oocyte complexes (COC) were collected from oviducts, and cumulus cells were removed with 0.1% hyaluronidase; 2) The oocytes of 13.5 h after hCG injection, with or without cumulus cells, were collected and cultured in HECM-3 for 5 and 10 h; 3) The COC collected from small vesicular follicles on the ovarian surface 72 h after eCG administration were cultured in HECM-3 supplemented with 5 μg mL–1 of FSH and 5 μg mL–1 of LH for 18 and 23 h. After the above treatments, denuded oocytes were stained with 10 μg mL–1 of Hoechst 33342 and observed on an inverted fluorescence microscope. During observation, the location of the FPB relative to the MII spindle was recorded as the angle (0–30°, 30–90°, 90–180°) between the line from the FPB to the center of the oocyte and the line from the spindle to the center of the oocyte. Oocytes were also stained with 10 μg mL–1 of propidium iodide (PI) before observation. The FPB that stained with PI were considered to be degenerated. All data were statistically analyzed by one-way ANOVA. Our results showed that 82.10% of FPB in oocytes collected at 13.5 h (n = 73) post-hCG were within the zone of 0–30°, which was significantly greater (P < 0.05) than those of oocytes collected at 18 h (n = 50; FPB 46.32%) and 23 h (n = 82; FPB 33.33%) post-hCG. The degenerate percentage of FPB in oocytes (without cumulus) cultured in vitro for 5 h (n = 72) and 10 h (n = 63) was 45 and 63%, respectively; this was significantly greater (P < 0.05) than that of oocytes with cumulus (5 h, n = 46; 32%; 10 h, n = 46; 45%). The percentage of FPB in oocytes matured in vitro for 18 h (n = 36) was 77.94% within the zone of 0–30°, which was significantly greater (P < 0.05) than the 38.83% seen in oocytes cultured in vitro for 23 h (n = 36). In conclusion, the results of this study demonstrate that a change in position of FPB relative to the MII oocyte chromosome is age-dependent in in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its change in position. In vitro-matured oocytes age more slowly than those of in vivo maturation and in vitro culture. These results define conditions for changing the FPB position relative to the MII oocyte chromosome and should facilitate the development of cloned golden hamsters as an animal model for human diseases.
98 HETEROCHROMATIN REPROGRAMMING IN MOUSE EARLY DEVELOPMENT
Authors: Wongtawan T. , Taylor J. , Wilmut I. , Pennings S.
Published: December 2008

Heterochromatin is essential for epigenetic gene silencing and nuclear chromatin architecture. Early mouse development is accompanied by dynamic epigenetic changes and heterochromatin restructuring in the cell nuclei of cleavage stage embryos. We have previously shown that disruption of heterochromatin markers such as DNA methylation following somatic nuclear transfer causes developmental failure (Beaujean N et al. 2004 Biol. Reprod. 71, 185–193). The aim of the present study was to investigate the transitions and maturation of heterochromatin during normal development to improve nuclear reprogramming technology and understand developmental abnormalities caused by epigenetic alterations in somatic nuclear transfer and assisted reproductive procedures. Mouse pre- and post-implantation embryos and ES cells from B6CBAF1 mice were used in experiments employing the following methodologies. Embryos and ES cells were stained by immunofluorescence to detect heterochromatin proteins and epigenetic markers. Images were captured using laser confocal microscopy. Relative quantification of fluorescent intensity was performed using Zeiss LSM-meta and WCIF-ImageJ software. mRNA of Histone methyltransferase (HMTase), suv39h, suv420h, ehmt, eset, and demethyltransferase (DHMTase), jmjd1a, jmjd2a, jmjd2c were quantified by real-time PCR. siRNA and specific inhibitors were used to study the function of suv39h, ehmt2, jmjd2c genes that may be responsible for heterochromatin reprogramming. Our results demonstrated that H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. The gene expression results showed the dynamic and temporal gene expression of HMTases and DHMTase during development. Inhibition of Ehmt2 and jmjd2c caused preimplantation developmental arrest. Furthermore, we found chromatin modification differences in the heterochromatin of ES cells, ICM and epiblast. We conclude that heterochromatin reprogramming might be essential for development because it may contribute to chromatin architecture, thus influencing gene expression. HMTases and DHTMases could be implicated in the mechanism of heterochromatin reprogramming. It is possible that Ehmt2 and jmjd2c play an important role in preimplantation development by modifying chromatin globally and at the local gene level.

This work was supported by a The Royal Thai Studentship. We acknowledge the contributions of technical assistance from T. O’Connor, B. Wongtawan and P. Travers.
99 EFFECT OF SUPPRESSION OF BOVINE DNA METHYLTRANSFERASE 1 ON EMBRYONIC DEVELOPMENT AND EXPRESSION OF IMPRINTED GENES
Authors: Wilaiphan P. , Rings F. , Hoelker M. , Tholen E. , Phatsara C. , Schellander K. , Tesfaye D.
Published: December 2008

DNA methyltransferase 1 (DNMT1) is believed to be involved in DNA methylation, which is a well-characterized epigenetic modulator shown to have essential functions in germ line and embryonic genome imprinting. This study was conducted to investigate the consequences of suppressing and inhibiting DNMT1 on the development, level of apoptosis, and expression of imprinted genes in pre-implantation bovine embryos. In vitro-produced zygotes were categorized into 4 groups; namely, those injected with Smartpool siRNA (SpsiRNA; Dharmacon Inc., Chicago, IL) (n = 800), 5aza-2′-deoxycytidine (5-AZA; Sigma, St. Louis, MO) (n = 864), nuclease-free water (n = 850), and uninjected control (n = 755). The mRNA expression data were generated using RT-PCR based on the relative standard curve method employing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a normalizer. Apoptotic index (API) was calculated by dividing the number of apoptotic cells to total cell number. The proportion of 2-, 4-, 8-cell and 2-, 4-, 8-, and 16-cell embryos was assessed 48 and 72 h, respectively, post-micro injection (pmi), whereas blastocyst rate was assessed at Day 8 pmi. Data on embryonic development and the relative mRNA abundance were analyzed using ANOVA followed by a multiple pair-wise mean comparison using Tukey test. The proportion of 2-, 4-, and 8-cell embryos at 48 h pmi was not significant among treatment groups. However, the proportion of the 8-cell embryos was significantly lower (P < 0.05) in SpsiRNA (16.3 ± 4.5) and 5-AZA injected groups (17.7 ± 4.9) compared with water-injected (26.8 ± 2.9) and uninjected controls (30.7 ± 6.2). The lowest total blastocyst rate (P < 0.05) was observed in the 5-AZA treatment group (16.9 ± 4.9) compared with SpsiRNA (23.4 ± 2.1) and water-injected (24.1 ± 5.3) and uninjected controls (29.4 ± 2.1). Microinjection of SpsiRNA reduced the target mRNA by 80 and 50% in 8-cell and blastocyst stage embryos, respectively, compared with uninjected control, and the protein expression level was also reduced at 8-cell embryos as confirmed by Western blotting. Injection of 5-AZA had no significant effect on mRNA or protein expression. The greatest API (P < 0.05) was found in SpsiRNA (4.2 ± 2.0) and 5-AZA (4.1 ± 1.7) injected groups compared with water-injected (2.8 ± 2.1) and uninjected controls (2.9 ± 2.3). The relative expression study also showed that microinjection of SpsiRNA and 5-AZA increased the expression of IGF2 (by 67 and 55%), IGF2R (29 and 30%), and IGFPB-4 (22 and 24%), respectively, compared with uninjected control, without affecting the expression of both IGF2R and IGFPB-4. In conclusion, suppression of DNMT1 resulted in lower proportion of 8-cell embryos, reduced blastocyst rate, and increased apoptotic index, and affected the expression of some imprinted genes, demonstrating a critical role of this gene in bovine embryonic development.
A DNA transposon-based approach to validate oncogenic mutations in the mouse
Authors: Su Qin , Prosser Haydn M. , Campos Lia S. , Ortiz Mariaestela , Nakamura Takuro , Warren Madhuri , Dupuy Adam J. , Jenkins Nancy A. , Copeland Neal G. , Bradley Allan , Liu Pentao
Published: December 2008

Large-scale cancer genome projects will soon be able to sequence many cancer genomes to comprehensively identify genetic changes in human cancer. Genome-wide association studies have also identified putative cancer associated loci. Functional validation of these genetic mutations
in vivo
is becoming a challenge. We describe here a DNA transposon-based platform that permits us to explore the oncogenic potential of genetic mutations in the mouse. Briefly, promoter-less human cancer gene cDNAs were first cloned into
Sleeping Beauty
(
SB
) transposons. DNA transposition in the mouse that carried both the transposons and the SB transposase made it possible for the cDNAs to be expressed from an appropriate endogenous genomic locus and in the relevant cell types for tumor development. Consequently, these mice developed a broad spectrum of tumors at very early postnatal stages. This technology thus complements the large-scale cancer genome projects.
Antagonistic selection from predators and pathogens alters food-web structure
Authors: Edeline Eric , Ari Tamara Ben , Vøllestad L. Asbjørn , Winfield Ian J. , Fletcher Janice M. , James J. Ben , Stenseth Nils C.
Published: December 2008

Selection can alter predator-prey interactions. However, whether and how complex food-webs respond to selection remain largely unknown. We show in the field that antagonistic selection from predators and pathogens on prey body-size can be a primary driver of food-web functioning. In Windermere, U.K., pike (
Esox lucius
, the predator) selected against small perch (
Perca fluviatilis
, the prey), while a perch-specific pathogen selected against large perch. The strongest selective force drove perch trait change and ultimately determined the structure of trophic interactions. Before 1976, the strength of pike-induced selection overrode the strength of pathogen-induced selection and drove a change to larger, faster growing perch. Predation-driven increase in the proportion of large, infection-vulnerable perch presumably favored the pathogen since a peak in the predation pressure in 1976 coincided with pathogen expansion and a massive perch kill. After 1976, the strength of pathogen-induced selection overrode the strength of predator-induced selection and drove a rapid change to smaller, slower growing perch. These changes made perch easier prey for pike and weaker competitors against juvenile pike, ultimately increasing juvenile pike survival and total pike numbers. Therefore, although predators and pathogens exploited the same prey in Windermere, they did not operate competitively but synergistically by driving rapid prey trait change in opposite directions. Our study empirically demonstrates that a consideration of the relative strengths and directions of multiple selective pressures is needed to fully understand community functioning in nature.
Dynein pulls microtubules without rotating its stalk
Authors: Ueno Hironori , Yasunaga Takuo , Shingyoji Chikako , Hirose Keiko
Published: December 2008

Dynein is a microtubule motor that powers motility of cilia and flagella. There is evidence that the relative sliding of the doublet microtubules is due to a conformational change in the motor domain that moves a microtubule bound to the end of an extension known as the stalk. A predominant model for the movement involves a rotation of the head domain, with its stalk, toward the microtubule plus end. However, stalks bound to microtubules have been difficult to observe. Here, we present the clearest views so far of stalks in action, by observing sea urchin, outer arm dynein molecules bound to microtubules, with a new method, “cryo-positive stain” electron microscopy. The dynein molecules in the complex were shown to be active in
in vitro
motility assays. Analysis of the electron micrographs shows that the stalk angles relative to microtubules do not change significantly between the ADP·vanadate and no-nucleotide states, but the heads, together with their stalks, shift with respect to their A-tubule attachments. Our results disagree with models in which the stalk acts as a lever arm to amplify structural changes. The observed movement of the head and stalk relative to the tail indicates a new plausible mechanism, in which dynein uses its stalk as a grappling hook, catching a tubulin subunit 8 nm ahead and pulling on it by retracting a part of the tail (linker).
The absence of reward induces inequity aversion in dogs
Authors: Range Friederike , Horn Lisa , Viranyi Zsófia , Huber Ludwig
Published: December 2008

One crucial element for the evolution of cooperation may be the sensitivity to others' efforts and payoffs compared with one's own costs and gains. Inequity aversion is thought to be the driving force behind unselfish motivated punishment in humans constituting a powerful device for the enforcement of cooperation. Recent research indicates that non-human primates refuse to participate in cooperative problem-solving tasks if they witness a conspecific obtaining a more attractive reward for the same effort. However, little is known about non-primate species, although inequity aversion may also be expected in other cooperative species. Here, we investigated whether domestic dogs show sensitivity toward the inequity of rewards received for giving the paw to an experimenter on command in pairs of dogs. We found differences in dogs tested without food reward in the presence of a rewarded partner compared with both a baseline condition (both partners rewarded) and an asocial control situation (no reward, no partner), indicating that the presence of a rewarded partner matters. Furthermore, we showed that it was not the presence of the second dog but the fact that the partner received the food that was responsible for the change in the subjects' behavior. In contrast to primate studies, dogs did not react to differences in the quality of food or effort. Our results suggest that species other than primates show at least a primitive version of inequity aversion, which may be a precursor of a more sophisticated sensitivity to efforts and payoffs of joint interactions.
The “Emblem of the Manifestation of the Iranian Spirit”: Hafiz and the Rise of the National Cult of Persian Poetry
Authors: Ferdowsi Ali
Published: December 2008

This is a study of the national sacralization of Hafiz as the “emblem of the manifestation of the Iranian spirit.” This sacralization began with E. G. Browne in 1902 but reached its definitive formulation in Abd al-Hosein Hazhir's monograph Hafiz-tashrih in 1928. For a nation to have a purchase on itself it must provide for a convocational experience. Shortly after the 1906 Constitutional Revolution, an ascending intellectual elite in Iran set out to do just that by fashioning a heterodox cult of the veneration of Persian poetry by recasting pre-modern texts, authors and their tombs into a national scripture authored by national prophets buried in sacred grounds which culminated in Hafiz as the “seal of its prophets.”
JAM-L–mediated leukocyte adhesion to endothelial cells is regulated in cis by α4β1 integrin activation
Authors: Luissint Anny-Claude , Lutz Pierre G. , Calderwood David A. , Couraud Pierre-Olivier , Bourdoulous Sandrine
Published: December 2008

Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L–VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.
Plasma membrane microdomains regulate turnover of transport proteins in yeast
Authors: Grossmann Guido , Malinsky Jan , Stahlschmidt Wiebke , Loibl Martin , Weig-Meckl Ina , Frommer Wolf B. , Opekarová Miroslava , Tanner Widmar
Published: December 2008

In this study, we investigate whether the stable segregation of proteins and lipids within the yeast plasma membrane serves a particular biological function. We show that 21 proteins cluster within or associate with the ergosterol-rich membrane compartment of Can1 (MCC). However, proteins of the endocytic machinery are excluded from MCC. In a screen, we identified 28 genes affecting MCC appearance and found that genes involved in lipid biosynthesis and vesicle transport are significantly overrepresented. Deletion of Pil1, a component of eisosomes, or of Nce102, an integral membrane protein of MCC, results in the dissipation of all MCC markers. These deletion mutants also show accelerated endocytosis of MCC-resident permeases Can1 and Fur4. Our data suggest that release from MCC makes these proteins accessible to the endocytic machinery. Addition of arginine to wild-type cells leads to a similar redistribution and increased turnover of Can1. Thus, MCC represents a protective area within the plasma membrane to control turnover of transport proteins.
Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis
Authors: Harris Kathryn P. , Tepass Ulrich
Published: December 2008

Cell rearrangements require dynamic changes in cell–cell contacts to maintain tissue integrity. We investigated the function of Cdc42 in maintaining adherens junctions (AJs) and apical polarity in the Drosophila melanogaster neuroectodermal epithelium. About one third of cells exit the epithelium through ingression and become neuroblasts. Cdc42-compromised embryos lost AJs in the neuroectoderm during neuroblast ingression. In contrast, when neuroblast formation was suppressed, AJs were maintained despite the loss of Cdc42 function. Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability. In addition, Cdc42 has a second function in regulating endocytotic trafficking, as it is required for the progression of apical cargo from the early to the late endosome. The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins. This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.
Binding interactions control SNARE specificity in vivo
Authors: Yang Hui-Ju , Nakanishi Hideki , Liu Song , McNew James A. , Neiman Aaron M.
Published: December 2008

Saccharomyces cerevisiae contains two SNAP25 paralogues, Sec9 and Spo20, which mediate vesicle fusion at the plasma membrane and the prospore membrane, respectively. Fusion at the prospore membrane is sensitive to perturbation of the central ionic layer of the SNARE complex. Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth. Suppression of the sporulation defect of an sso1 mutant requires expression of a chimeric form of Spo20 carrying the SNARE helices of Sec9. Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells. This mutant form of Spo20 displayed enhanced activity in in vitro fusion assays, as well as tighter binding to Sso1 and Snc2. These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.
How Can We Strengthen the Evidence Base in Public Health Preparedness?
Authors: Nelson Christopher D. , Beckjord Ellen Burke , Dausey David J. , Chan Edward , Lotstein Debra , Lurie Nicole
Published: December 2008

The lack of frequent real-world opportunities to study preparedness for large-scale public health emergencies has hindered the development of an evidence base to support best practices, performance measures, standards, and other tools needed to assess and improve the nation’s multibillion dollar investment in public health preparedness. In this article, we argue that initial funding priorities for public health systems research on preparedness should focus on using engineering-style methods to identify core preparedness processes, developing novel data sources and measures based on smaller-scale proxy events, and developing performance improvement approaches to support the translation of research into practice within the wide variety of public health systems found in the nation. (Disaster Med Public Health Preparedness. 2008;2:247–250)
The habenular nuclei: a conserved asymmetric relay station in the vertebrate brain
Authors: Bianco Isaac H , Wilson Stephen W
Published: December 2008

The dorsal diencephalon, or epithalamus, contains the bilaterally paired habenular nuclei and the pineal complex. The habenulae form part of the dorsal diencephalic conduction (DDC) system, a highly conserved pathway found in all vertebrates. In this review, we shall describe the neuroanatomy of the DDC, consider its physiology and behavioural involvement, and discuss examples of neural asymmetries within both habenular circuitry and the pineal complex. We will discuss studies in zebrafish, which have examined the organization and development of this circuit, uncovered how asymmetry is represented at the level of individual neurons and determined how such left–right differences arise during development.
HBEGF, SRA1, and IK: Three cosegregating genes as determinants of cardiomyopathy
Authors: Friedrichs Frauke , Zugck Christian , Rauch Gerd-Jörg , Ivandic Boris , Weichenhan Dieter , Müller-Bardorff Margit , Meder Benjamin , El Mokhtari Nour Eddine , Regitz-Zagrosek Vera , Hetzer Roland , Schäfer Arne , Schreiber Stefan , Chen Jian , Neuhaus Isaac , Ji Ruiru , Siemers Nathan O. , Frey Norbert , Rottbauer Wolfgang , Katus Hugo A. , Stoll Monika
Published: December 2008

Human dilated cardiomyopathy (DCM), a disorder of the cardiac muscle, causes considerable morbidity and mortality and is one of the major causes of sudden cardiac death. Genetic factors play a role in the etiology and pathogenesis of DCM. Disease-associated genetic variations identified to date have been identified in single families or single sporadic patients and explain a minority of the etiology of DCM. We show that a 600-kb region of linkage disequilibrium (LD) on 5q31.2-3, harboring multiple genes, is associated with cardiomyopathy in three independent Caucasian populations (combined P-value = 0.00087). Functional assessment in zebrafish demonstrates that at least three genes, orthologous to loci in this LD block, HBEGF, IK, and SRA1, result independently in a phenotype of myocardial contractile dysfunction when their expression is reduced with morpholino antisense reagents. Evolutionary analysis across multiple vertebrate genomes suggests that this heart failure-associated LD block emerged by a series of genomic rearrangements across amphibian, avian, and mammalian genomes and is maintained as a cluster in mammals. Taken together, these observations challenge the simple notion that disease phenotypes can be traced to altered function of a single locus within a haplotype and suggest that a more detailed assessment of causality can be necessary.
Phase I study of TZT‐1027, a novel synthetic dolastatin 10 derivative and inhibitor of tubulin polymerization, given weekly to advanced solid tumor patients for 3 weeks
Authors: Yamamoto Nobuyuki , Andoh Masahiro , Kawahara Masaaki , Fukuoka Masahiro , Niitani Hisanobu
Published: December 2008

TZT‐1027 is a novel synthetic dolastatin 10 derivative that inhibits tubulin polymerization. A phase I study was conducted to determine the maximum tolerated dose (MTD) of TZT‐1027, and to assess its pharmacokinetic profile in Japanese patients with advanced solid tumors following administration of the drug weekly for 3 weeks. Eligible patients had advanced solid tumors that failed to respond to standard therapy or for which no standard therapy was available, and met the following criteria: performance status ≤2 and acceptable organ function. The MTD was defined as the highest dose at which more than two‐thirds of the patients experienced grade 4 hematological toxicity or grade 3/4 non‐hematological toxicity during weekly TZT‐1027 administration for 3 weeks. Forty patients were enrolled in the present study. Twelve doses between 0.3 and 2.1 mg/m2 were evaluated. Grade 4 neutropenia was the principal dose‐limiting toxicity (DLT). At a dose of 2.1 mg/m2, two patients developed DLT: one patient developed grade 4 neutropenia, grade 3 myalgia, and grade 4 constipation, and the other one developed grade 4 neutropenia and grade 3 constipation. At a dose level of 1.8 mg/m2, toxicity was acceptable and no DLT was observed. The area under the curve and maximum concentration of TZT‐1027 tended to increase linearly with the dose. The DLT observed were neutropenia, myalgia, and constipation, and the MTD was 2.1 mg/m2. The recommended dose for a phase II study was determined to be 1.8 mg/m2 for the drug administered weekly for 3 weeks. (Cancer Sci 2009; 100: 316–321)
Transforming growth factor‐β signaling and cancer: The 28th Sapporo Cancer Seminar, 25–27 June 2008
Authors: Miyazono Kohei
Published: December 2008

The Sapporo Cancer Seminars have been held annually since 1981. The 28th Sapporo Cancer Seminar was held on 26–27 June 2008 at the Hokkaido University Conference Hall, focusing on transforming growth factor (TGF)‐β signaling and cancer. More than 150 scientists participated in the seminar, and it provided a great deal of information on the role of TGF‐β signaling in carcinogenesis and tumor metastasis. The possible use of TGF‐β antagonists for treatment of cancers was also discussed at the seminar. (Cancer Sci 2009; 100: 363–365)
Human neutrophils switch to an activated phenotype after homing to the lung irrespective of inflammatory disease
Authors: Fortunati E , Kazemier K M , Grutters J C , Koenderman L , Van den Bosch Van J M M
Published: December 2008

Systemic inflammation can be investigated by changes in expression profiles of neutrophil receptors. Application of this technology for analysis of neutrophil phenotypes in diseased tissues is hampered by the absence of information regarding the modulation of neutrophil phenotypes after extravasation to tissues under non-inflammatory conditions. To fill this gap we measured the expression of neutrophil receptors in bronchoalveolar lavage fluid (BALF) and in the peripheral blood of healthy volunteers, which included both smokers and non-smokers. Blood and BALF neutrophils were identified by CD16bright/CD45dim cells, and triple-stained with antibodies directed against integrins, chemokine- and Fcγ-receptors. BALF neutrophils of healthy volunteers showed an activated phenotype characterized by Mac-1 (CD11b)bright, L-selectin (CD62L)dim, intrecellular adhesion molecule 1 (ICAM-1) (CD54)bright, FcγRII (CD32)bright, C5a receptor (CD88)bright and CD66bbright. A similar phenotype was observed for BALF neutrophils of patients affected by sarcoidosis. Furthermore, our results demonstrate a modulated expression of C5a receptor (CD88) and ICAM-1 (CD54) in neutrophils of sarcoidosis patients. In conclusion, our data indicate that neutrophils found in the lung exhibit an activated phenotype under both homeostatic and inflammatory conditions.
Macroinvertebrates as indicators of fish absence in naturally fishless lakes
Authors: SCHILLING EMILY GAENZLE , LOFTIN CYNTHIA S. , HURYN ALEXANDER D.
Published: December 2008

1. Little is known about native communities in naturally fishless lakes in eastern North America, a region where fish stocking has led to a decline in these habitats.

2. Our study objectives were to: (i) characterise and compare macroinvertebrate communities in fishless lakes found in two biophysical regions of Maine (U.S.A.): kettle lakes in the eastern lowlands and foothills and headwater lakes in the central and western mountains; (ii) identify unique attributes of fishless lake macroinvertebrate communities compared to lakes with fish and (iii) develop a method to efficiently identify fishless lakes when thorough fish surveys are not possible.

3. We quantified macroinvertebrate community structure in the two physiographic fishless lake types (n = 8 kettle lakes; n = 8 headwater lakes) with submerged light traps and sweep nets. We also compared fishless lake macroinvertebrate communities to those in fish‐containing lakes (n = 18) of similar size, location and maximum depth. We used non‐metric multidimensional scaling to assess differences in community structure and t‐tests for taxon‐specific comparisons between lakes.

4. Few differences in macroinvertebrate communities between the two physiographic fishless lake types were apparent. Fishless and fish‐containing lakes had numerous differences in macroinvertebrate community structure, abundance, taxonomic composition and species richness. Fish presence or absence was a stronger determinant of community structure in our study than differences in physical conditions relating to lake origin and physiography.

5. Communities in fishless lakes were more speciose and abundant than in fish‐containing lakes, especially taxa that are large, active and free‐swimming. Families differing in abundance and taxonomic composition included Notonectidae, Corixidae, Gyrinidae, Dytiscidae, Aeshnidae, Libellulidae and Chaoboridae.

6. We identified six taxa unique to fishless lakes that are robust indicators of fish absence: Graphoderus liberus, Hesperocorixa spp., Dineutus spp., Chaoborus americanus, Notonecta insulata and Callicorixa spp. These taxa are collected most effectively with submerged light traps.

7. Naturally fishless lakes warrant conservation, because they provide habitat for a unique suite of organisms that thrive in the absence of fish predation.
Identifying abuse among women: use of clinical guidelines by nurses and midwives
Authors: Svavarsdottir Erla Kolbrun , Orlygsdottir Brynja
Published: December 2008

Title.  Identifying abuse among women: use of clinical guidelines by nurses and midwives.

Aim.  This aim of this study to identify the incidence of violence against women seeking healthcare services and evaluate the use of clinical guidelines to identify interpersonal violence.

Methods.  A cross‐sectional survey was carried out. Data were collected over a period of 7 months in 2005 and 2006. The samples consisted of 14 nurses, 10 midwives and 208 women in Iceland (101 women visiting the Emergency Department and 107 receiving prenatal care at the High Risk Prenatal Care Clinic).

Results.  Twenty women (19·6%) who visited the Emergency Department and 21 women (19·8%) who came to the High Risk Prenatal Care Clinic had been sexually abused at some point in their lives by close family members. Within the preceding 12 months, 18 women at the Emergency Department (19·1%) and eight at the High Risk Prenatal Care Clinic (7·5%) reported physical abuse, and 22 women (22·2%) at the Emergency Department and 12 (11·5%) at the High Risk Prenatal Care Clinic reported emotional abuse. A majority of the nurses and midwives indicated that the guidelines were efficient for assessing/screening for gender violence in emergency and high risk clinical settings.

Conclusion.  Screening for abuse of women at emergency and high risk clinics is crucial, not only to offer the women the immediate interventions they might need, but also to ensure the future provision of appropriate healthcare services.
Independent evolution of functional Pm3 resistance genes in wild tetraploid wheat and domesticated bread wheat
Authors: Yahiaoui Nabila , Kaur Navreet , Keller Beat
Published: December 2008

The Pm3 alleles of cultivated bread wheat confer gene for gene resistance to the powdery mildew fungus. They represent a particular case of plant disease resistance gene evolution, because of their recent origin and possible evolution after the formation of hexaploid wheat. The Pm3 locus is conserved in tetraploid wheat, thereby allowing the comparative evolutionary study of the same resistance locus in a domesticated species and in one of its wild ancestors. We have identified 61 Pm3 allelic sequences from wild and domesticated tetraploid wheat subspecies. The Pm3 sequences corresponded to 24 different haplotypes. They showed low sequence diversity, differing by only a few polymorphic sequence blocks that were further reshuffled between alleles by gene conversion and recombination. Polymorphic sequence blocks are different from the blocks found in functional Pm3 alleles of hexaploid wheat, indicating an independent evolution of the Pm3 loci in the two species. A new functional gene was identified in a wild wheat accession from Syria. This gene, Pm3k, conferred intermediate race‐specific resistance to powdery mildew, and consists of a mosaic of gene segments derived from non‐functional alleles. This demonstrates that Pm3‐based resistance is not very frequent in wild tetraploid wheat, and that the evolution of functional resistance genes occurred independently in wild tetraploid and bread wheat. The Pm3 sequence variability and geographic distribution indicated that diversity was higher in wild emmer wheat from the Levant area, compared with the accessions from Turkey. Further screens for Pm3 functional genes in wild wheat should therefore focus on accessions from the Levant region.