bluuebunny/crossref_metadata_2025 huggingface dataset:
Recovery of deformation substructure and coarsening of particles on annealing severely plastically deformed Al–Mg–Si alloy and analysis of strengthening mechanisms
Authors: Gutierrez-Urrutia I. , Muñoz-Morris M.A. , Morris D.G.
Published: February 2006

An Al–Mg–Si alloy was annealed to various solutionized and aged states and was then severely plastically deformed by equal channel angular pressing (ECAP). These materials were subsequently annealed for a range of times and temperatures to induce precipitation, dislocation recovery, and grain growth, with changes of mechanical behavior followed by tensile testing. Precipitation of excess solute was seen to occur in all cases, independent of the initial heat treated state, but the particles present appear to play only a small role in stabilizing the deformation substructure, at least until significant particle and grain coarsening has occurred, when discontinuous grain coarsening can be provoked. The strength of materials is examined, and the respective contributions of loosely arranged dislocations, many grain boundaries, and dispersed particles are deduced. It is shown that dislocation strengthening is significant in as-deformed, as well as lightly annealed materials, with grain boundary strengthening providing the major contribution thereafter.
Genetic Diversity among Hatchery Stocks and Established Populations of Rainbow Trout in Missouri
Authors: Dillman Casey B. , Koppelman Jeffrey B.
Published: February 2006

Introduction of rainbow trout Oncorhynchus mykiss in Missouri over the last 125 years has established numerous self‐sustaining populations. While many populations have been repeatedly stocked, some have received few fish beyond the original releases, and it is possible that those populations currently represent their source lineages. There is angler interest in fishing “wild” rainbow trout populations both for historical purposes and for the fighting ability of hooked fish. A genetic inventory was conducted to determine the extent to which Missouri's wild fish differ from hatchery stocks. Haplotype diversity in the mitochondrial DNA (mtDNA) cytochrome b gene and allele frequencies at four microsatellite loci were examined. Significant differences in mtDNA genotypes were found among several wild and hatchery sources, indicating that multiple sources of rainbow trout were probably represented. Crane Creek—long considered a stronghold of McCloud River (California) redband trout O. mykiss stonei in Missouri—and its tributary, Spring Creek, were characterized as having a large proportion of unique haplotypes. Microsatellite diversity was largely shared among individuals within populations, and average heterozygosity was high. By including this information in decisions about regulations and stocking, the Missouri Department of Conservation can manage rainbow trout to satisfy angler interests with respect to both historical and performance‐related population characteristics.
Picturing Meaning
Authors: Romano Tom
Published: February 2006

Teachers can help students develop mental picture‐making skills by reading aloud to them. But they must do more than read fluently: Teachers must orally interpret texts. The voice is an instrument to aid meaning making. By projecting the voice, varying the pitch, altering reading speed, inserting strategic pauses, and enunciating words, teachers can make written language live. To become strong readers, students must learn to complete such transactions when they read silently, and teachers' skilled oral interpretations can support that development and demonstrate possibilities for how written voices might sound.
Crafting Questions That Address Comprehension Strategies in Content Reading
Authors: Fordham Nancy W.
Published: February 2006

This article describes initial attempts by preservice and inservice teachers enrolled in university reading courses to devise questions that encourage students to read content texts strategically. Teachers at both levels tended to confuse questions that encourage the use of comprehension strategies during reading with questions that assess comprehension of what has been read. Despite understanding the aspects of cognition that promote good comprehension, at first these elementary and middle‐grade teachers appeared to revert to ingrained questioning models that largely ignored the cognitive components of comprehension.

The distinction between the design and functions of these questions must be clear to teachers if they are to be effective in helping students construct meaning from subject matter texts. University classes that provide modeling and practice in crafting and asking strategic questions support content teachers in this important skill.
Tales Out of School: Accounting for Adolescents in a Literacy Reform Community
Authors: Brozo William
Published: February 2006

Guided by the hypothesis that students are most directly affected by—but least often consulted about—educational policy, practice, and reform, the author gathered interview data from students at a high school that was enacting new schoolwide literacy initiatives. In spite of the reforms, student achievement on state tests remained unchanged from previous years.

The interview data support the assertion that the success or failure of literacy reform efforts depends in large measure on the cooperation and investment of students. Findings suggest further that adolescents, like their teachers, are not compliant functionaries but critical negotiators of change. On the basis of issues raised in the interviews, the author offers suggestions for ensuring all students are authorized as legitimate members of reform communities. Otherwise, the author claims, literacy innovations, which are often perceived as worthwhile by those lauding and enacting them, may have a limited degree of success in improving youths' academic performance.
Mission Accomplished…It's Learnable Now: Voices of Mature Challenged Spellers Using a Word Study Approach
Authors: Massengill Donita
Published: February 2006

The purpose of this study was to evaluate the impact of an instructional spelling approach, Word Study, on adult learners' spelling. The author sought to learn more about participants' stage of spelling development, to gain information from the adults on their interpretations of this approach, and to determine if a short‐term intervention would change their spelling or self‐perception.

Nine adult participants were administered one reading and two spelling pre‐ and postassessments. Eight participants were in the syllables‐and‐affixes stage, and one possessed adequate orthographic knowledge to be placed in the derivational relations stage. The nine participants received individualized Word Study intervention (word sorts) for 12 sessions between pre‐ and posttests. The adults made significant spelling gains on their knowledge of word features as measured by the Upper Level Spelling Inventory; however, their improvement in raw scores was not significant. Further, the participants demonstrated an increase in confidence, self‐esteem, and willingness to attempt the spelling of unknown words. All nine participants favorably viewed Word Study as a respectable spelling approach for adults. A section is devoted to implications for practice.
Activin A Maintains Self-Renewal and Regulates Fibroblast Growth Factor, Wnt, and Bone Morphogenic Protein Pathways in Human Embryonic Stem Cells
Authors: Xiao Lei , Yuan Xuan , Sharkis Saul J.
Published: February 2006

Human embryonic stem cells (hESCs) self-renew indefinitely while maintaining pluripotency. The molecular mechanism underlying hESCs self-renewal and pluripotency is poorly understood. To identify the signaling pathway molecules that maintain the proliferation of hESCs, we performed a microarray analysis comparing an aneuploid H1 hESC line (named H1T) versus euploid H1 hESC line because the H1T hESC line demonstrates a self-renewal advantage while maintaining pluripotency. We find differential gene expression for the Nodal/Activin, fibroblast growth factor (FGF), Wnt, and Hedgehog (Hh) signaling pathways in the H1T line, which implicates each of these molecules in maintaining the undifferentiated state, whereas the bone morphogenic protein (BMP) and Notch pathways could promote hESCs differentiation. Experimentally, we find that Activin A is necessary and sufficient for the maintenance of self-renewal and pluripotency of hESCs and supports long-term feeder and serum-free growth of hESCs. We show that Activin A induces the expression of Oct4, Nanog, Nodal, Wnt3, basic FGF, and FGF8 and suppresses the BMP signal. Our data indicates Activin A as a key regulator in maintenance of the stemness in hESCs. This finding will help elucidate the complex signaling network that maintains the hESC phenotype and function.
Cellular Memory and Hematopoietic Stem Cell Aging
Authors: Kamminga Leonie M. , de Haan Gerald
Published: February 2006

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation in order to sustain lifelong blood production and simultaneously maintain the HSC pool. However, there is clear evidence that HSCs are subject to quantitative and qualitative exhaustion. In this review, we briefly discuss several known aspects of the stem cell aging process, including DNA damage, telomere shortening, and oxidative stress. Besides these known players, there is increasing evidence that higher order chromatin structure, largely defined by the histone code and affecting transcriptional activity, is important. A model is suggested which describes how epigenetic regulation of gene transcription by modulation of the chromatin structure in stem cells can account for regulation of the aging program.
Improved Safety of Hematopoietic Transplantation with Monkey Embryonic Stem Cells in the Allogeneic Setting
Authors: Shibata Hiroaki , Ageyama Naohide , Tanaka Yujiro , Kishi Yukiko , Sasaki Kyoko , Nakamura Shinichiro , Muramatsu Shin-ichi , Hayashi Satoshi , Kitano Yoshihiro , Terao Keiji , Hanazono Yutaka
Published: February 2006

Cynomolgus monkey embryonic stem cell (cyESC)-derived in vivo hematopoiesis was examined in an allogeneic transplantation model. cyESCs were induced to differentiate into the putative hematopoietic precursors in vitro, and the cells were transplanted into the fetal cynomolgus liver at approximately the end of the first trimester (n = 3). Although cyESC-derived hematopoietic colony-forming cells were detected in the newborns (4.1%–4.7%), a teratoma developed in all newborns. The risk of tumor formation was high in this allogeneic transplantation model, given that tumors were hardly observed in immunodeficient mice or fetal sheep that had been xeno-transplanted with the same cyESC derivatives. It turned out that the cyESC-derived donor cells included a residual undifferentiated fraction positive for stage-specific embryonic antigen (SSEA)-4 (38.2% ± 10.3%) despite the rigorous differentiation culture. When an SSEA-4-negative fraction was transplanted (n = 6), the teratoma was no longer observed, whereas the cyESC-derived hematopoietic engraftment was unperturbed (2.3%–5.0%). SSEA-4 is therefore a clinically relevant pluripotency marker of primate embryonic stem cells (ESCs). Purging pluripotent cells with this surface marker would be a promising method of producing clinical progenitor cell preparations using human ESCs.
Identification of Label-Retaining Cells in Mouse Endometrium
Authors: Chan Rachel W.S. , Gargett Caroline E.
Published: February 2006

Human and mouse endometrium (lining of the uterus) undergo cycles of growth and regression as part of each reproductive cycle. A well-known method to identify somatic stem/progenitor cells and their location in the stem cell niche is the label-retaining cell (LRC) approach. We hypothesized that mouse endometrium contains small populations of both epithelial and stromal somatic stem/progenitor cells that may be detected by the LRC technique. The overall objective of this study was to identify and quantify LRCs in mouse endometrium, to determine their location, and to identify their niche in this highly regenerative tissue. Endometrium was labeled for 3 days with bromodeoxyuridine (BrdU) in postnatal day 3 (P3) mice prior to gland development and prepubertal (P19) mice after glands had formed, followed by chase periods of up to 12 weeks. After an 8-week chase, 3% of epithelial nuclei immunostained with BrdU antibody and were considered epithelial LRCs. These were primarily located in the luminal epithelium. Epithelial LRCs did not express estrogen receptor-α (ER-α). Stromal LRCs (6%) were found adjacent to luminal epithelium, at the endometrial-myometrial junction, and near blood vessels after a 12-week chase. Stromal LRCs were stem cell antigen-1, CD45−, and some (16%) expressed ER-α, indicating their capacity to respond to estrogen and transmit paracrine signals to epithelial cells for endometrial epithelium regeneration. Both epithelial LRCs and some stromal LRCs, mainly located at the endometrial-myometrial junction, were recruited into the cell cycle after estrogen-stimulated endometrial regeneration, indicating a functional response to proliferative signals. This study has demonstrated for the first time the presence of both epithelial and stromal LRCs in mouse endometrium, suggesting that these stem-like cells may be responsible for endometrial regeneration.
Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary
Authors: Slot Karin A , Voorendt Marsha , de Boer-Brouwer Mieke , van Vugt Harmke H , Teerds Katja J
Published: February 2006

In the present investigation, the localization of proteins involved in ovarian apoptosis were studied throughout the estrous cycle in the presence of fluctuating hormone levels. Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA expression and proteins were detected in all ovarian tissue extracts, though the amount of protein varied with the phase of the estrous cycle. Fas, Bax and caspase-3 protein levels were highest at diestrus and decreased thereafter towards metestrus. In contrast, Fas ligand and Bcl-2 protein levels were lowest at diestrus and increased toward metestrus. Immunohistochemistry revealed that the staining of the anti-apoptotic protein Bcl-2 was more pronounced in healthy preantral follicles than in atretic follicles. In contrast, the pro-apoptotic proteins Fas, Fas ligand, Bax and active caspase-3 were more predominantly present in atretic follicles. In the ovarian surface epithelium (OSE), Fas, procaspase-3 and Bcl-2 immunostaining appeared independent of the phase of the estrous cycle. Fas ligand and Bax staining was detected particularly during proestrus in OSE cells surrounding the ovulatory follicles, while active caspase-3 was observed only in OSE cells at the postovulatory site during estrus. The proportion of luteal cells that stained positively for Fas, Bax and caspase-3 increased with the age of the corpus luteum, while Fas ligand and Bcl-2 immunostaining was strongest in newly formed corpora lutea and decreased thereafter. In conclusion, the components of the Fas signalling pathway were differentially expressed throughout the estrous cycle in a variety of ovarian cell types, which may correspond to hormone dependent survival mechanisms.
A 36 residues insertion in the dimerization domain of the growth hormone receptor results in defective trafficking rather than impaired signaling
Authors: Maamra M , Milward A , Esfahani H Zarkesh , Abbott L P , Metherell L A , Savage M O , Clark A J L , Ross R J M
Published: February 2006

Growth hormone insensitivity syndrome (GHIS) has been reported in a family homozygous for a point mutation in the GH receptor (GHR) that activates an intronic pseudoexon. The resultant GHR (GHR1–656) includes a 36 amino-acids insertion after residue 207, in the region known to be important for homodimerization of GHR. We have examined the functional consequences of such an insertion in mammalian cells transfected with the wild type (GHRwt) and mutated GHR (GHR1–656). Radio-ligand binding and flow cytometry analysis showed that GHR1–656 is poorly expressed at the cell surface compared with GHRwt. Total membrane binding and Western blot analysis showed no such difference in the level of total cellular GHR expressed for GHR1–656 vs GHRwt. Immunofluorescence showed GHR1–656 to have different cellular distribution to the wild type receptor (GHRwt), with the mutated GHR being mainly perinuclear and less vesicular than GHRwt. Western blot analysis showed GH-induced phosphorylation of Jak2 and Stat5 for both GHR1–656 and GHRwt, although reduced Stat5 activity was detected with GHR1–656, consistent with lower levels of expression of GHR1–656 than GHRwt at the cell surface. In conclusion, we report that GHIS, due to a 36 amino-acids insertion in the extracellular domain of GHR, is likely to be explained by a trafficking defect rather than by a signalling defect of GHR.
Changes in α-estradiol receptor and progesterone receptor expression in the locus coeruleus and preoptic area throughout the rat estrous cycle
Authors: Helena Cleyde Vanessa Vega , de Oliveira Poletini Maristela , Sanvitto Gilberto Luiz , Hayashi Shinji , Franci Celso Rodrigues , Anselmo-Franci Janete Aparecida
Published: February 2006

We have previously shown that the locus coeruleus (LC) is essential for triggering surges of LH. Since LC neurons are responsive to estradiol, which induces progesterone receptor (PR) expression, this study aimed to investigate whether LC neurons express the α-estradiol receptor (αER) and PR as well as comparing such responses to that observed in the preoptic area (POA). Female rats were perfused at 10, 14 and 16 h on each day of the estrous cycle, and a blood sample was collected for estradiol, progesterone and LH measurements. αER- and PR immunoreactive (ir) neurons were detected in POA and LC by immunocytochemistry (ICC). Higher plasma estradiol levels were observed on the day of proestrus, when a smaller number of αER-ir POA neurons were detected. An increase in the number of αER-ir neurons were observed at 16 h of proestrus and estrus. The number of PR-ir neurons increased in POA only at 16 h of proestrus, and remained unchanged during all other days and times. The profile of αER-ir and PR-ir neurons in LC changed over the estrous cycle, with a lower expression on metestrus morning and reaching a peak on diestrus afternoon before declining on the day of proestrus. However, on estrus afternoon, αER-ir neurons increased, while PR-ir neurons decreased which may be related to the prolactin surge of estrus. These data show that LC neurons express αER and PR and seem to be more sensitive to variations in estradiol than POA. Also, the fluctuation in αER and PR observed for LC neurons seems to accompany the hormonal events that occur during the estrous cycle. This profile of αER and PR expression might be related to the ability of estradiol and progesterone in regulating the activity of LC neurons, which could be associated to the control mechanisms of LH and prolactin release.
The pro-opiomelanocortin genes in rainbow trout (Oncorhynchus mykiss): duplications, splice variants, and differential expression
Authors: Leder E H , Silverstein J T
Published: February 2006

Pro-opiomelanocortin (POMC) is a precursor for several important peptide hormones involved in a variety of functions ranging from stress response to energy homeostasis. In mammals and fish, the POMC-derived peptide α-melanocyte stimulating hormone (MSH) is known to be involved in appetite suppression through its interaction with melanocortin-4 receptors. The details of energy homeostasis in fishes are beginning to be elucidated and many of the genes involved in mammalian neuroendocrine signaling pathways are being discovered in fish. In salmonid fishes such as the rainbow trout, genome duplication adds another degree of complexity when trying to compare gene function and homology with other vertebrates. This is true of the POMC gene. Two copies of the POMC gene were previously identified, A and B, presumably resulting from the salmonid duplication. However, while investigating POMC involvement in the feeding response of rainbow trout, a second copy of POMC-A was discovered which is more likely the result of the salmonid duplication and suggests that POMC-B is a duplicate resulting from the earlier teleost duplication prior to tetrapod divergence. The duplicated POMC-A had five deleted amino acids, five inserted amino acids, and 39 amino acid differences from the published POMC-A. In addition to the duplicate POMC-A, a splice variant of the published POMC-A sequence was also identified. Quantitative real-time PCR assays were developed for the different POMC transcripts, and expression was examined in a variety of tissues. Expression of POMC transcripts was highest in the pituitary for all POMC genes, but varied among other tissues for POMC-A1, POMC-A2, POMC-A2s, and POMC-B. POMC-A1 was the only transcript to respond significantly to food deprivation.
The G972R variant of the insulin receptor substrate-1 gene impairs insulin signaling and cell differentiation in 3T3L1 adipocytes; treatment with a PPARγ agonist restores normal cell signaling and differentiation
Authors: Sentinelli F , Filippi E , Cavallo M G , Romeo S , Fanelli M , Baroni M G
Published: February 2006

The insulin receptor substrate-1 (IRS-1) plays a central role in insulin sensitivity, and association studies have shown that the IRS-1 G972R variant is a risk factor for insulin resistance. However, how this mutation may lead to impaired insulin sensitivity is still to be determined. Our study aimed to evaluate, after transfection of the IRS-1 G972R variant in 3T3L1 adipocytes, the effect of this mutation on insulin signaling and on cell differentiation. The 3T3L1 cells were transfected with pcDNA3 expression vector containing either the human wild-type IRS-1 or the G972R variant. After induction of differentiation, the 3T3L1 transfected with wild-type IRS-1 differentiated in 6–8 days, while the cells transfected with G972R variant did not differentiate. To determine whether the defect in IRS-1 was responsible for this, we analyzed the expression of several genes involved in the insulin signaling pathway. Results showed that PPARγ expression was significantly reduced in cells transfected with the mutated IRS-1, together with a significant decrease in binding of phosphatidylinositol-3 kinase (PI 3-kinase) to IRS-1 G972R and in PI 3-kinase activity. In addition, we observed that the interaction between the insulin receptor (IR) and the IRS-1 G972R protein was increased and that the autophosphorylation of the IR was significantly inhibited in 3T3L1-G972R cells compared with 3T3L1-WT. Treatment of the 3T3L1-G972R cells with pioglitazone (PIO), a PPARγ agonist, restored differentiation with higher level of PPARγ expression and restoration of PI 3-kinase binding to IRS-1 G972R and PI 3-kinase activity. IR autophosphorylation was also increased. Withdrawal of PIO in fully differentiated 3T3L1-G972R cells determined the reappearance of the insulin signaling defect. Finally, we observed higher levels of IRS-2 expression, suggesting that IRS-2 may play a more important role in adipocyte insulin signaling. In conclusion, IRS-1 G972R variant impairs insulin signaling, and treatment with PPARγ agonist restores the normal phenotype of 3T3L1 cells.
Locally produced estrogen promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation and decreased apoptosis
Authors: Chagin A S , Chrysis D , Takigawa M , Ritzen E M , Sävendahl L
Published: February 2006

The importance of estrogens for the regulation of longitudinal bone growth is unequivocal. However, any local effect of estrogens in growth plate cartilage has been debated. Recently, several enzymes essential for estrogen synthesis were shown to be expressed in rat growth plate chondrocytes. Local production of 17β-estradiol (E2) has also been demonstrated in rat costal chondrocytes. We aimed to determine the functional role of locally produced estrogen in growth plate cartilage. The human chondrocyte-like cell line HCS-2/8 was used to study estrogen effects on cell proliferation (3H-labeled thymidine uptake) and apoptosis (cell death detection ELISA kit). Chondrocyte production of E2 was measured by RIA and organ cultures of fetal rat metatarsal bones were used to study the effects of estrogen on longitudinal growth rate. We found that significant amounts of E2 were produced by HCS-2/8 chondrocytes (64.1 ± 5.3 fmol/3 days/106cells). The aromatase inhibitor letrozole (1 μM) and the pure estrogen receptor antagonist ICI 182,780 (10 μM) inhibited proliferation of HCS-2/8 chondrocytes by 20% (P<0.01) and almost 50% (P<0.001), respectively. Treatment with ICI 182,780 (10 μM) increased apoptosis by 228% (P<0.05). Co-treatment with either caspase-3 or pan-caspase inhibitors completely blocked ICI 182,780-induced apoptosis (P<0.001 vs ICI 182,780 only). Moreover, both ICI 182,780 (10 μM) and letrozole (1 μM) decreased longitudinal growth of fetal rat metatarsal bones after 7 days of culture (P<0.01). In conclusion, our data clearly show that chondrocytes endogenously produce E2 and that locally produced estrogen stimulates chondrocyte proliferation and protects from spontaneous apoptosis. In addition, longitudinal growth is promoted by estrogens locally produced within the epiphyseal growth plate.
Gonadotropin-secreting cells in ovariectomized rats treated with different oestrogen receptor ligands: a modulatory role for ERβ in the gonadotrope?
Authors: Sánchez-Criado J E , de las Mulas J Martín , Bellido C , Navarro V M , Aguilar R , Garrido-Gracia J C , Malagón M M , Tena-Sempere M , Blanco A
Published: February 2006

In the rat, oestrogen is a key regulator of gonadotrophin synthesis and release through activation of oestrogen receptors (ERs). Gonadotropes express α and β isoforms of ER and both can activate transcription in response to oestrogen. These experiments were aimed at evaluating the relative contribution of ERα and ERβ on gonadotrope morphology, progesterone receptor (PR) expression and LH secretion. Ovariectomized rats were daily injected over 3 days with 25 μg oestradiol benzoate, 0.3 or 1.5 mg of the selective ERα agonist propylpyrazole triol (PPT) with or without 1.5, 3.0 or 4.5 mg of the selective ERβ agonist diarylpropionitrile (DPN), DPN alone, and 0.3 or 3 mg of tamoxifen. Controls were given 0.2 ml oil. Serum concentration and pituitary content of LH, gonadotrope PR expression, pituitary PR content, and gonadotrope morphology were analyzed by RIA, immunohistochemistry, Western blotting and light and electron microscopy, respectively. Results showed that PPT reversed all consequences of ovariectomy, DPN mimicked the effects of PPT except for its LH-releasing action and tamoxifen had ERα-like responses. When combined with PPT, DPN attenuated ERα effects without interfering with its LH-releasing activity. Oestradiol benzoate had similar effects to those of combined PPT and DPN. It is suggested that (i) the structural reorganization of the cytoplasmic organelles provided by oestrogen, and the shrinkage of the ovariectomy-induced hypertrophy of gonadotropes, which precedes the expression of PR, are evoked by ERα and modulated, in a ying–yang fashion, by ERβ; and (ii) the oestrogen-dependent exocytosis of LH, the final step in the secretory process, is dependent on ERα exclusively.
Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R
Authors: Utriainen Pauliina , Liu Jianqi , Kuulasmaa Tiina , Voutilainen Raimo
Published: February 2006

Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2′deoxycytidine (Azad; 0.1–100 μM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.
Both N-terminal and C-terminal regions of steroid sulfatase are important for enzyme activity
Authors: Sugawara T , Nomura E , Hoshi N
Published: February 2006

Steroid sulfatase (STS) is localized in the endoplasmic reticulum and catalyzes desulfation of 3β-hydroxysteroid sulfates. X-linked ichthyosis (XLI) is an inherited skin disorder caused by deficiency of STS enzyme activity. We previously reported a case in which XLI with a one-base change in the STS gene and variation in amino acid Q560P developed. In this study, we performed molecular analysis to determine the importance of terminal regions of STS and the effect of mutant STS on STS enzyme activity. To examine the effect of terminal truncated STS on the enzyme activity, N- and C-terminal truncated STS expression vectors were transfected into COS-1 cells. The activity of truncated STS lacking the N-terminal regions declined, and the activity of C-terminal-truncated STS declined with extension of the truncated C-terminal region. Although the results of pulse-chase experiments showed that a one-base mutant STS (Q560P) and C-terminal-truncated STS (ΔC2 (1–559)) had no effects on protein synthesis and degradation, the mutant STS and C-terminal-truncated STS have dominant negative effect on STS enzyme activity when the STS mutant or truncated STS protein and a wild-type STS protein coexist in cells. Results of coprecipitation of the truncated STS with an STS–FLAG fusion protein showed that STS formed a dimer conformation in cells. In this study, we have shown that both the N-terminal region and C-terminal region are important for STS enzyme activity. The C-terminal mutant has a dominant negative effect on wild-type STS.
Suppressive effects of dehydroepiandrosterone and the nuclear factor-κB inhibitor parthenolide on corticotroph tumor cell growth and function in vitro and in vivo
Authors: Taguchi T , Takao T , Iwasaki Y , Nishiyama M , Asaba K , Hashimoto K
Published: February 2006

Dehydroepiandrosterone (DHEA) is believed to have an anti-tumor effect, as well as anti-inflammatory, antioxidant, and anti-aging effects. To clarify the possible inhibitory action of DHEA on pituitary tumor cells, we tested the effects of DHEA, alone or in combination with the nuclear factor-κB (NF-κB) inhibitor parthenolide (PRT), on AtT20 corticotroph cell growth and function both in vitro and in vivo. We found that, in vitro, DHEA and PRT had potent inhibitory effects on pro-opiomelanocortin and NF-κB-dependent gene expression. They also suppressed the transcription activity of survivin, a representative anti-apoptotic factor, and induced apoptosis in this cell line. Furthermore, using BALB/C nude mice with xenografts of AtT20 cells in vivo, we found that the combined administration of DHEA and PRT significantly attenuated tumor growth and survivin expression. The treatment also decreased the elevated plasma corticosterone levels and ameliorated the malnutrition induced by tumor growth. Altogether, these results suggested that combined treatments of DHEA and PRT potently inhibit the growth and function of corticotroph tumor cells both in vitro and in vivo. This effect may, at least partly, be caused by the suppressive effects of these compounds, such as survivin and other inhibitor of apoptosis proteins, on NF-κB-mediated gene transcription.
Diabetes causes marked changes in function and metabolism of rat neutrophils
Authors: Alba-Loureiro T C , Hirabara S M , Mendonça J R , Curi R , Pithon-Curi T C
Published: February 2006

Several studies have shown impairment of neutrophil function, a disorder that contributes to the high incidence of infections in diabetes. Since glucose and glutamine play a key role in neutrophil function, we investigated their metabolism in neutrophils obtained from the peritoneal cavity of streptozotocin-induced diabetic rats. The activities of hexokinase, glucose-6-phosphate dehydrogenase (G6PDH), phosphofructokinase (PFK), citrate synthase, phosphate-dependent glutaminase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were assayed. Glucose, glutamine, lactate, glutamate and aspartate, and the decarboxylation of [U-14C], [1-14C] and [6-14C]glucose; [U-14C]palmitic acid; and [U-14C]glutamine were measured in 1-h incubated neutrophils. Phagocytosis capacity and hydrogen peroxide (H2O2) production were also determined. All measurements were carried out in neutrophils from control, diabetic and insulin-treated (2–4IU/day) diabetic rats. Phagocytosis and phorbol myristate acetate (PMA)-stimulated H2O2 production were decreased in neutrophils from diabetic rats. The activities of G6PDH and glutaminase were decreased, whereas that of PFK was raised by the diabetic state. The activities of the remaining enzymes were not changed. Diabetes decreased the decarboxylation of [1-14C]glucose and [U-14C]glutamine; however, [6-14C]glucose and [U-14C]palmitic acid decarboxylation was increased. These observations indicate that changes in metabolism may play an important role in the impaired neutrophil function observed in diabetes. The treatment with insulin abolished the changes induced by the diabetic state even with no marked change in glycemia. Therefore, insulin may have a direct effect on neutrophil metabolism and function.
Differential expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and activin type-I and type-II receptors in preovulatory and prehierarchical follicles of the laying hen ovary
Authors: Lovell T M , Knight P G , Gladwell R T
Published: February 2006

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-β (TGFβ) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (~40 mm).

Ovaries were collected (n=16) from mid-sequence hens maintained on a long-day photoschedule (16 h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of mRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0.05) in 8–9.9 mm follicles, whereas ActRIIA rose significantly from 6–7.9 mm to 8–9.9 mm, before falling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan mRNA expression rose 3-fold from 4–5.9 mm to 8–9.9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4–7.9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0.05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 mm to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActRIIA increased 2-fold from F2 to F1 (P < 0.05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before falling ~50% in the F1.

In all follicles studied expression of betaglycan and ActRI (granulosa: r=0.65, P < 0.001, n=144/group; theca: r=0.49, P < 0.001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI, ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11.4-fold; ActRIIB, 5.1-fold; ActRI, 3.8-fold; ActRIIA, 2.8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.
Region-specific expression of nitric oxide synthases in the bovine oviduct during the oestrous cycle and in vitro
Authors: Ulbrich S E , Rehfeld S , Bauersachs S , Wolf E , Rottmayer R , Hiendleder S , Vermehren M , Sinowatz F , Meyer H H D , Einspanier R
Published: February 2006

Nitric oxide synthases (NOS) account for the endogenous production of nitric oxide (NO), a small and permeable bioreactive molecule. NO is known to act as a paracrine mediator during various processes associated with female reproduction. In the present study, the mRNA expression of the endothelial (eNOS) and inducible (iNOS) NO synthases were examined in bovine oviduct epithelial cells (BOEC) during the oestrous cycle. In addition, eNOS and iNOS mRNA and protein were localised by in situ hybridisation and immunocytochemistry respectively. Furthermore, the effects of exogenously applied oestradiol-17β and progesterone on NOS mRNA regulation were studied in a suspension culture of BOEC. The eNOS mRNA abundance was low around ovulation (day 0) and increased significantly until pro-oestrus (day 18) in the ampulla. Immunoreactive protein of eNOS was detected predominantly in endothelial cells as well as in secretory oviduct epithelial cells at pro-oestrus. The iNOS mRNA concentration was significantly reduced in the isthmus at pro-oestrus (day 18) and oestrus (day 0) compared with persistently high levels in the ampulla. By in situ hybridisation, specific iNOS transcripts were additionally demonstrated in the oviduct epithelium. Immunoreactive iNOS protein was localised in secretory epithelial cells as well as in the lamina muscularis. The in vitro stimulation showed that both NOS were stimulated by progesterone, but not by oestradiol-17β. The region-specific modulated expression of eNOS and iNOS provides evidence for an involvement of endogenously produced NO in the regulation of oviductal functions.
Phosphoinositide-derived messengers in endocrine signaling
Authors: Balla T
Published: February 2006

One of the fundamental questions in endocrinology is how circulating or locally produced hormones affect target cell functions by activating specific receptors linked to numerous signal-transduction pathways. An important subset of G protein-coupled cell-surface receptors can activate phospholipase C enzymes to hydrolyze a small but critically important class of phospholipids, the phosphoinositides. Although this signaling pathway has been extensively explored over the last 20 years, this has proven to be only the tip of the iceberg, and the multiplicity and diversity of the cellular functions controlled by phosphoinositides have surpassed any imagination. Phosphoinositides have been found to be key regulators of ion channels and transporters, and controllers of vesicular trafficking and the transport of lipids between intracellular membranes. Essentially, they organize the recruitment and regulation of signaling protein complexes in specific membrane compartments. While many of these processes have been classically studied by cell biologists, molecular endocrinology cannot ignore these recent advances, and now needs to integrate the cell biologist’s views in the modern concept of how hormones affect cell functions and how derailment of simple molecular events can lead to complex endocrine and metabolic disorders.
Upflow Reactors for Riparian Zone Denitrification
Authors: van Driel Peter W. , Robertson William D. , Merkley L. Craig
Published: February 2006

We used permeable reactive subsurface barriers consisting of a C source (wood particles), with very high hydraulic conductivities (∼0.1–1 cm s−1), to provide high rates of riparian zone NO3–N removal at two field sites in an agricultural area of southwestern Ontario. At one site, a 0.73‐m3 reactor containing fine wood particles was monitored for a 20‐mo period and achieved a 33% reduction in mean influent NO3–N concentration of 11.5 mg L−1 and a mean removal rate of 4.5 mg L−1 d−1 (0.7 g m−2 d−1). At the second site, four smaller reactors (0.21 m3 each), two containing fine wood particles and two containing coarse wood particles, were monitored for a 4‐mo period and were successful in attenuating mean influent NO3–N concentrations of 23.7 to 35.1 mg L−1 by 41 to 63%. Mean reaction rates for the two coarse‐particle reactors (3.2 and 7.8 mg L−1 d−1, or 1.5 and 3.4 g m−2 d−1) were not significantly different (p > 0.2) than the rates observed in the two fine‐particle reactors (5.0 and 9.9 mg L−1 d−1, or 1.8–3.5 g m−2 d−1). A two‐dimensional ground water flow model is used to illustrate how permeable reactive barriers such as these can be used to redirect ground water flow within riparian zones, potentially augmenting NO3− removal in this environment.
Predicting Soil Erosion for Alternative Land Uses
Authors: Wang Erda , Xin Chang , Williams Jimmy R. , Xu Cheng
Published: February 2006

The APEX (Agricultural Policy–Environmental eXtender) model developed in the United States was calibrated for northwestern China's conditions. The model was then used to investigate soil erosion effects associated with alternative land uses at the ZFG (Zi‐Fang‐Gully) watershed in northwestern China. The results indicated that the APEX model could be calibrated reasonably well (±15% errors) to fit those areas with >50% slope within the watershed. Factors being considered during calibration include runoff, RUSLE (Revised Universal Soil Loss Equation) slope length and steepness factor, channel capacity flow rate, floodplain saturated hydraulic conductivity, and RUSLE C factor coefficient. No changes were made in the APEX computer code. Predictions suggest that reforestation is the best practice among the eight alternative land uses (the status quo, all grass, all grain, all grazing, all forest, half tree and half grass, 70% tree and 30% grain, and construction of a reservoir) for control of water runoff and soil erosion. Construction of a reservoir is the most effective strategy for controlling sediment yield although it does nothing to control upland erosion. For every 1 Mg of crop yield, 11 Mg of soil were lost during the 30‐yr simulation period, suggesting that expanding land use for food production should not be encouraged on the ZFG watershed. Grass species are less effective than trees in controlling runoff and erosion on steep slopes because trees generally have deeper and more stable root systems.
Increased Wind Erosion from Forest Wildfire: Implications for Contaminant‐Related Risks
Authors: Whicker Jeffrey J. , Pinder John E. , Breshears David D.
Published: February 2006

Assessments of contaminant‐related human and ecological risk require estimation of transport rates, but few data exist on wind‐driven transport rates in nonagricultural systems, particularly in response to ecosystem disturbances such as forest wildfire and also relative to water‐driven transport. The Cerro Grande wildfire in May of 2000 burned across ponderosa pine (Pinus ponderosa Douglas ex P.&C. Lawson var. scopulorum Englem.) forest within Los Alamos National Laboratory in northern New Mexico, where contaminant transport and associated post‐fire inhalation risks are of concern. In response, the objectives of this study were to measure and compare wind‐driven horizontal and vertical dust fluxes, metrics of transport related to wind erosion, for 3 yr for sites differentially affected by the Cerro Grande wildfire: unburned, moderately burned (fire mostly confined to ground vegetation), and severely burned (crown fire). Wind‐driven dust flux was significantly greater in both types of burned areas relative to unburned areas, by more than one order of magnitude initially and by two to three times 1 yr after the fire. Unexpectedly, the elevated dust fluxes did not decrease during the second and third years in burned areas, apparently because ongoing drought delayed post‐fire recovery. Our estimates enable assessment of amplification in contaminant‐related risks following a major type of disturbance‐wildfire, which is expected to increase in intensity and frequency due to climate change. More generally, our results highlight the importance of considering wind‐ as well as water‐driven transport and erosion, particularly following disturbance, for ecosystem biogeochemistry in general and human and ecological risk assessment in particular.
Spatial Analysis of Land Use Impact on Ground Water Nitrate Concentrations
Authors: Benson Victoria S. , VanLeeuwen John A. , Sanchez Javier , Dohoo Ian R. , Somers George H.
Published: February 2006

In spatial analyses of causes or health effects of environmental pollutants, small units of analyses are usually preferred for internal environmental homogeneity reasons but can only be done when fine resolution data are available for most units. Objectives of this study were to determine which land use practices were spatially associated with ground water nitrate concentrations across Prince Edward Island (PEI), Canada, and which spatial aggregation is the preferred unit of analyses. Nitrate concentrations were determined for 4855 samples from private wells. Validated field‐by‐field land use data were available. Average nitrate concentration and percentage of area for the 14 major land use categories in PEI were determined for each of three spatial aggregations: watersheds based on topography and hydrology; freeform polygon boundaries based on similar neighboring nitrate concentrations; and 500‐m buffer zones around each well. Results showed that the percentages of potato, grain, and hay coverage were positive predictors of ground water nitrate concentrations. Percentage of blueberry was a marginally significant negative predictor in the watershed and freeform polygon models, and percentage of residential coverage was a positive predictor in the freeform polygon and buffer zone models. Spatial autocorrelation was present in the freeform polygon and buffer zone models even after land use was taken into account. In conclusion, analyses based on watersheds produced the best predictive model with the percentages of land cover of potato, hay, and grain being significantly associated with ground water nitrate concentrations, and the percentages of blueberry, clear‐cut woodland, and other agriculture being marginally significant.