bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell cycle checkpoints and their impact on anticancer therapeutic strategies
Authors: Eastman Alan
Published: January 2004

Cells contain numerous pathways designed to protect them from the genomic instability or toxicity that can result when their DNA is damaged. The p53 tumor suppressor is particularly important for regulating passage through G1 phase of the cell cycle, while other checkpoint regulators are important for arrest in S and G2 phase. Tumor cells often exhibit defects in these checkpoint proteins, which can lead to hypersensitivity; proteins in this class include ataxia–telangiectasia mutatated (ATM), Meiotic recanbination 11 (Mre11), Nijmegen breakage syndrome 1 (Nbs 1), breast cancer susceptibility genes 1 and 2 (BRCA1), and (BRCA2). Consequently, tumors should be assessed for these specific defects, and specific therapy prescribed that has high probability of inducing response. Tumors defective in p53 are frequently considered resistant to apoptosis, yet this defect also provides an opportunity for targeted therapy. When their DNA is damaged, p53‐defective tumor cells preferentially arrest in S or G2 phase where they are susceptible to checkpoint inhibitors such as caffeine and UCN‐01. These inhibitors preferentially abrogate cell cycle arrest in p53‐defective cells, driving them through a lethal mitosis. Wild type p53 can prevent abrogation of arrest by elevating levels of p21waf1 and decreasing levels of cyclins A and B. During tumorigenesis, tumor cells frequently loose checkpoint controls and this facilitates the development of the tumor. However, these defects also represent an Achilles heel that can be targeted to improve current therapeutic strategies. © 2003 Wiley‐Liss, Inc.
Platelet‐derived growth factor‐BB phosphorylates heat shock protein 27 in cardiac myocytes
Authors: Takenaka Motoki , Matsuno Hiroyuki , Ishisaki Akira , Nakajima Keiichi , Hirade Kouseki , Takei Mariko , Yasuda Eisuke , Akamatsu Shigeru , Yoshimi Naoki , Kato Kanefusa , Kozawa Osamu
Published: January 2004

It is recognized that heat shock protein 27 (HSP27) is highly expressed in heart. In the present study, we investigated whether platelet‐derived growth factor (PDGF) phosphorylates HSP27 in mouse myocytes, and the mechanism underlying the HSP27 phosphorylation. Administration of PDGF‐BB induced the phosphorylation of HSP27 at Ser‐15 and ‐85 in mouse cardiac muscle in vivo. In primary cultured myocytes, PDGF‐BB time dependently phosphorylated HSP27 at Ser‐15 and ‐85. PDGF‐BB stimulated the phosphorylation of p44/p42 mitogen‐activated protein (MAP) kinase, p38 MAP kinase, and stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) among the MAP kinase superfamily. SB203580, a specific inhibitor of p38 MAP kinase, reduced the PDGF‐BB‐stimulated phosphorylation of HSP27 at both Ser‐15 and ‐85, and phosphorylation of p38 MAP kinase. However, PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK, failed to affect the HSP27 phosphorylation. These results strongly suggest that PDGF‐BB phosphorylates HSP27 at Ser‐15 and ‐85 via p38 MAP kinase in cardiac myocytes. © 2003 Wiley‐Liss, Inc.
PPARγ1 synthesis and adipogenesis in C3H10T1/2 cells depends on S‐phase progression, but does not require mitotic clonal expansion
Authors: Cho Young C. , Jefcoate Colin R.
Published: January 2004

Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I), dexamethasone (D), and methylisobutylxanthine (M), administered with a fresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator‐activated receptor γ1 (PPARγ1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that mitotic clonal expansion of 3T3‐L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S‐phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPARγ1 synthesis and lipogenesis. Different S‐phase inhibitors (aphidicolin, hydroxyurea, l‐mimosine, and roscovitin) each arrested cells in S‐phase, under hormonal stimulation, and completely blocked PPARγ1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPARγ1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M‐phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one‐half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPARγ was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPARγ1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. Importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non‐differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S‐phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPARγ synthesis and subsequent adipocyte differentiation. © 2003 Wiley‐Liss, Inc.
Breast cancer cells induce osteoblast apoptosis: A possible contributor to bone degradation
Authors: Mastro Andrea M. , Gay Carol V. , Welch Danny R. , Donahue Henry J. , Jewell Jennifer , Mercer Robyn , DiGirolamo Douglas , Chislock Elizabeth M. , Guttridge Kristin
Published: January 2004

Breast cancer cells exhibit a predilection for metastasis to bone. There, the metastases usually bring about bone loss with accompanying pain and loss of function. One way that breast cancer cells disrupt the normal pattern of bone remodeling is by activating osteoclasts, the bone degrading cells. Nevertheless, targeting the osteoclasts does not cure the disease or result in bone repair. These observations indicate that osteoblast function also may be compromised. The objective of this study was to investigate the interaction of metastatic breast cancer cells with osteoblasts. Human metastatic breast cancer cells, MDA‐MB‐435 or MDA‐MB‐231, or their conditioned media were co‐cultured with a human osteoblast line hFOB1.19. The breast cancer cells caused an increase in the prevalence of apoptotic osteoblasts. Apoptotic osteoblasts detected by the TUNEL assay or by caspase activity increased approximately two to fivefold. This increase was not seen with non‐metastatic MDA‐MB‐468 cells. In an investigation of the mechanism, it was determined that the hFOB1.19 cells expressed fas and that fas was functional. Likewise the hFOB1.19 cells were susceptible to TNF‐α, but this cytokine was not detected in the conditioned medium of the breast cancer cells. This study indicates that osteoblasts are the target of breast cancer cell‐induced apoptosis, but fas/fas‐ligand and TNF‐α, two common initiators of cell death, are probably not involved in this aspect of the metastases/bone cell axis. There are several mechanisms that remain to be explored in order to determine how breast cancer cells bring about osteoblast apoptosis. Even though the specific initiator of apoptosis remains to be identified, the results of this study suggest that the mechanism is likely to be novel. © 2003 Wiley‐Liss, Inc.
Regulatory factor X2 (RFX2) binds to the H1t/TE1 promoter element and activates transcription of the testis‐specific histone H1t gene
Authors: Wolfe Steven A. , Wilkerson Donald C. , Prado Susan , Grimes Sidney R.
Published: January 2004

Transcription of the mammalian testis‐specific linker histone H1t gene occurs only in pachytene primary spermatocytes during spermatogenesis. Studies of the wild type (Wt) and mutant H1t promoters in transgenic mice show that transcription of the H1t gene is dependent upon the TE promoter element. We purified an 85 kDa protein from rat testis nuclear extracts using the TE1 subelement as an affinity chromatography probe and analysis revealed that the protein was RFX2. The TE1 element is essentially an X‐box DNA consensus element and regulatory factor X (RFX) binds specifically to this element. Polyclonal antibodies directed against RFX2 supershift the low mobility testis nuclear protein complex formed in electrophoretic mobility shift assays (EMSA). RFX2 derived from primary spermatocytes, where the transcription factor is relatively abundant, binds with high affinity to the TE1 element. Coexpression of RFX2 together with an H1t promoter/reporter vector activates the H1t promoter in a cultured GC‐2spd germinal cell line, but mutation of either the TE1 subelement or the TE2 subelements represses activity. These observations lead us to conclude that the TE1 and TE2 subelements of the testis‐specific histone H1t promoter are targets of the transcription factor RFX2 and that this factor plays a key role in activating transcription of the H1t gene in primary spermatocytes. Published 2003 Wiley‐Liss, Inc.
Differentially up‐regulated genes in proliferating porcine neonatal pancreas cells caused by epidermal growth factor
Authors: Jeon Sung Yoon , Baek Kwang‐Hyun , Kim Yong‐Soo , Park Chung‐Gyu , Kwon Hyuk Sang , Ko Seung Hyun , Song Ki‐Ho , Yoo Soon Jib , Son Hyun Shik , Cha Bong Yun , Lee Kwang Woo , Son Ho Young , Kang Sung Koo , Yoon Kun‐Ho
Published: January 2004

Pancreatic duct cells are considered to be a major source for β‐cell regeneration or neogenesis. Although epidermal growth factor (EGF) is a well‐known important growth factor for pancreas development, the control of pancreatic duct cell growth and differentiation by EGF is poorly understood. In this study, we focused on identifying the genes that were differentially up‐regulated in response to EGF stimulation using monolayer cultured porcine neonatal pancreas cells. Cells were obtained from 1 to 3 day old pigs, dispersed and cultured for 8 days. Monolayer cultured porcine pancreas cells were comprised of duct cells and some endocrine and mesenchymal cells (75.2 ± 15.1, 19.6 ± 4.9, and 9.5 ± 3.1%, respectively). After 16 h in serum free media, cells were treated with 100 μg/L EGF for 24 h. Differentially expressed genes were screened by subtractive hybridization. 3H‐thymidine uptake was significantly increased by EGF with time (untreated vs. 24 h treated, untreated vs. 48 h treated: 305.5 ± 3.5 cpm vs. 380.3 ± 17.3 cpm (P < 0.05), 309.2 ± 4.51 vs. 929 ± 9.19 cpm, (P < 0.005), respectively). Three hundred and fify cDNA clones were obtained by subtractive hybridization and the inserts were confirmed in 161 colonies and then sequenced. Finally, we found increased mRNA expression of five unknown and five known genes, including cytochrome c oxidase subunit I (COI), cyclooxygenase‐2 (COX‐2), matrix metalloproteinase‐13 (MMP‐13), Wiskott–Aldrich syndrome protein interacting protein (WASPIP), and hyaluronan synthase‐2 (HAS‐2). We confirmed the up‐regulation of these genes by Northern blot and semi‐quantitative RT‐PCR at various time points. The present findings opened new targets for the research on the mechanisms of pancreatic duct cell proliferation by EGF. © 2003 Wiley‐Liss, Inc.
Constitutive and inducible expression of cytochromes P4501A (CYP1A1 and CYP1A2) in normal prostate and prostate cancer cells
Authors: Sterling Kenneth M. , Cutroneo Kenneth R.
Published: January 2004

Constitutive and benzo[a]pyrene (B[a]P) inducible expression of CYP1A1 and CYP1A2 in prostate cancer and normal prostate epithelial cells were examined by immunoblotting. Androgen independent prostate cancer cell lines DU145 and PC3 have constitutive expression of CYP1A and CYP1A1 and CYP1A2, respectively. Four micromolar B[a]P did not appear to induce CYP1A1 or CYP1A2 expression in DU145 or PC3 cells. The androgen dependent prostate cancer cell line, LnCap, also has constitutive expression of CYP1A1 and CYP1A2. However, both CYP1A1 and CYP1A2 are induced by treatment of LnCap cells with 4 μM B[a]P. Untreated normal prostate and primary prostate tumor cells have no detectable CYP1A1 expression. Treatment with 4 μM B[a]P induced CYP1A1 expression in both normal and primary tumor prostate cells. Constitutive CYP1A2 expression was detected in normal prostate cells with little or no induction by exposure to 4 μM B[a]P. Primary prostate tumor cells did not show constitutive expression of CYP1A2. However, CYP1A2 was induced by 4 μM B[a]P in primary prostate tumor cells. These observations indicate that hormonal and cancer specific factors affect the expression and induction of the phase I metabolic enzymes, CYP1A1 and CYP1A2 in prostate cells. These observations may be related to the potential smoking‐linked higher risk of prostate cancer development and morbidity of prostate cancer patients who smoke. © 2003 Wiley‐Liss, Inc.
Cleavage of p130Cas in anoikis
Authors: Wei Lin , Yang Yu , Zhang Xing , Yu Qiang
Published: January 2004

p130Cas is a multifunctional signaling adaptor protein. It integrates and relays signals generated from a variety of extracellular stimuli and regulates a number of cellular activities including cell death. In this study, we analyzed the regulation and function of p130Cas in anoikis, a type of apoptosis caused by disruption of cell‐matrix interactions. We found that p130Cas was specifically cleaved during anoikis in anoikis‐sensitive epithelial cells, but not in anoikis‐resistant tumor cells. There is a close correlation between p130Cas cleavage and anoikis. Furthermore, we found that the cleavage of p130Cas, as well as another focal adhesion component FAK, is different from that of caspase substrate PARP and spectrin. Although caspases and calpain were found to be involved in the cleavage of p130Cas, there appear to be other unidentified proteases that are mainly responsible for the cleavage of p130Cas, particularly at the early stage of anoikis. Overexpression of the p130Cas cleavage product induced apoptosis. Taken together, these data suggest that there are novel proteases involved in the cleavage of p130Cas during anoikis, which may be functionally involved in the onset of anoikis. p130Cas may have a dual role in the regulation of anoikis. On one hand, it mediates a survival signal from cell‐matrix interactions when cells are attached to the extracellular matrix. On the other hand, it participates in executing cell death when cell‐matrix interactions are disrupted. These observations provide new insights into the understanding of the function of p130Cas and the molecular mechanism of anoikis. © 2003 Wiley‐Liss, Inc.
Mediating of caspase‐independent apoptosis by cadmium through the mitochondria‐ROS pathway in MRC‐5 fibroblasts
Authors: Shih Chwen‐Ming , Ko Wun‐Chang , Wu Jui‐Sheng , Wei Yau‐Huei , Wang Leng‐Fang , Chang E‐E. , Lo Tsui‐Yun , Cheng Huey‐Hwa , Chen Chien‐Tsu
Published: January 2004

Cadmium (Cd) is an environmental pollutant of global concern with a 10–30‐year biological half‐life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 μM Cd induces MRC‐5 cells, normal human lung fibroblasts, to undergo caspase‐independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis‐inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl‐Val‐Ala‐Asp‐(ome) fluoromethyl ketone (Z‐VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase‐independent apoptosis at concentrations varied from 25 to 150 μM, which was modulated by reactive oxygen species (ROS) scavengers, such as N‐acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9‐fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl‐X‐rosamine (CMXRos) and an anti‐AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h‐treatment) was a prelude to the translocation of caspase‐independent pro‐apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC‐5 fibroblasts, Cd induced caspase‐independent apoptosis through a mitochondria‐ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase‐independent cell death triggered by Cd. © 2003 Wiley‐Liss, Inc.
Angiostatin‐induced inhibition of endothelial cell proliferation/apoptosis is associated with the down‐regulation of cell cycle regulatory protein cdk5
Authors: Sharma Meena R. , Tuszynski George P. , Sharma Mahesh C.
Published: January 2004

Endothelial cells (ECs) are quiescent in normal blood vessels, but undergo rapid bursts of proliferation after vascular injury, hypoxia or induced by powerful angiogenic cytokines like fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Deregulated proliferation of ECs facilitates angiogenic processes and promotes tumor growth. In dividing cells, cell cycle‐associated protein kinases, which are referred as cyclin‐dependent kinases (cdks), regulate proliferation, differentiation, senescence, and apoptosis. Cyclin‐dependent kinase‐5 (cdk5) is expressed in neuronal cells and plays an important role in neurite outgrowth, of neuronal migration and neurogenesis, its functions in non‐neuronal cells are unclear. Here, we show for the first time that the cdk5 is expressed at high levels in proliferating bovine aortic endothelial (BAE) cells, by contrast insignificant low levels of cdk5 expression in quiescent BAE cells. In addition, bFGF up‐regulates cdk5 expression in a dose‐dependent fashion. Interestingly, temporal expression data suggests that cdk5 expression is very low between 24–48 h, but high level of cdk5 expression was detected during 60–72 h. This later time corresponds to the time of completion of one cell cycle (doubling of cell population) of BAE cell culture. Angiostatin (AS), a powerful inhibitor of angiogenesis inhibits ECs proliferation in dose‐dependent manner with concomitant down‐regulation of cdk5 expression. The role of cdk5 in ECs, proliferation and apoptosis was confirmed by selective inhibition of cdk5 expression by the purine derivative roscovitine, which inhibits bFGF‐stimulated BAE cells proliferation and induces apoptosis in dose‐specific manner. By contrast, the roscovitine analog olomoucine, which is a specific inhibitor of cdk4, but not of cdk5 failed to affect ECs proliferation and apoptosis. These data suggest for the first time that neuron specific protein cdk5 may have significant role in the regulation of ECs proliferation, apoptosis, and angiogenesis and extends beyond its role in neurogenesis. © 2003 Wiley‐Liss, Inc.
Nuclear microenvironments support assembly and organization of the transcriptional regulatory machinery for cell proliferation and differentiation
Authors: Stein Gary S. , Lian Jane B. , van Wijnen Andre J. , Stein Janet L. , Javed Amjad , Montecino Martin , Zaidi S. Kaleem , Young Daniel , Choi Je‐Yong , Gutierrez Soraya , Pockwinse Shirwin
Published: January 2004

The temporal and spatial organization of transcriptional regulatory machinery provides microenvironments within the nucleus where threshold concentrations of genes and cognate factors facilitate functional interactions. Conventional biochemical, molecular, and in vivo genetic approaches, together with high throughput genomic and proteomic analysis are rapidly expanding our database of regulatory macromolecules and signaling pathways that are requisite for control of genes that govern proliferation and differentiation. There is accruing insight into the architectural organization of regulatory machinery for gene expression that suggests signatures for biological control. Localized scaffolding of regulatory macromolecules at strategic promoter sites and focal compartmentalization of genes, transcripts, and regulatory factors within intranuclear microenvironments provides an infrastructure for combinatorial control of transcription that is operative within the three dimensional context of nuclear architecture. © 2003 Wiley‐Liss, Inc.
Gene array identification of osteoclast genes: Differential inhibition of osteoclastogenesis by cyclosporin A and granulocyte macrophage colony stimulating factor
Authors: Day Christopher J. , Kim Michael S. , Stephens Sébastien R.J. , Simcock Wendy E. , Aitken Cathy J. , Nicholson Geoff C. , Morrison Nigel A.
Published: January 2004

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) stimulates the formation of multinucleate osteoclast‐like cells. Treatment with M‐CSF alone results in the formation of macrophage‐like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R∝), transcription factors (nuclear factor of activated T‐cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPα)), and ribosomal proteins (60S L17 and 40S S20). Real‐time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c‐Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate‐resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30‐fold. Constant exposure of M‐CSF‐ and RANKL‐treated cells to GM‐CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis. © 2004 Wiley‐Liss, Inc.
Physiology of hibernation inRana pipiens: metabolic rate, critical oxygen tension, and the effects of hypoxia on several plasma variables
Authors: Ultsch Gordon R. , Reese Scott A. , Stewart E. Ray
Published: January 2004

Rates of O2consumption (V̇O2) were determined for adult northern leopard frogs (Rana pipiens) submerged at 3 °C at water PO2s (PwO2) ranging from 0–160 mmHg. The critical O2tension (Pc) was 36.4 mmHg. Hematocrit and blood levels of PO2, glucose, lactate, pH, [Na+], [K+], and osmolality were determined for frogs submerged for two days. Above a PwO2of 50 mmHg, blood PO2ranged from 1–7 mmHg, which was sufficient to allow the frogs to function entirely aerobically. Plasma [lactate] increased as PwO2fell below 50 mmHg, the increase preceding significant changes in any other variable, and apparently preceding a fall in V̇O2. Most other variables showed little or no change from those of air‐breathing control animals, even during anoxia. We present an analysis of the importance of a large decrease in Pcin permitting frogs to successfully overwinter in icebound ponds and of the factors that contribute to that decrease.J. Exp. Zool. 301A:169–176, 2004.© 2004 Wiley Liss, Inc.
Forelimb spike regeneration in Xenopus laevis: Testing for adaptiveness
Authors: Tassava Roy A.
Published: January 2004

Experiments were designed to test adaptability of forelimb spike regenerates in Xenopus laevis froglets. The results show that when amputation is at the radius/ulna level, regeneration occurs in 100% of the cases and a single spike of cartilage is the result. The spike regenerates originating from radius/ulna level amputations can be used for feeding and froglet growth is only minimally compromised by the spike. The spike grows in length as the froglet body grows and thus is in homeostasis with the body. The spike develops nuptial pad tissue in reproductively mature males and is occasionally molted, indicating responsiveness to gonadal and thyroid hormones. Finally, and most important, the spike can be used for amplexus and successful mating. In contrast, spikes originating from humerus level amputations were considerably shorter and regeneration from that limb level was less frequent. When amputation was at the body wall regeneration did not occur. J. Exp. Zool. 301A:150–159, 2004. © 2004 Wiley‐Liss, Inc.
Lymphoid aggregates in gonads of embryos, hatchlings, and young of turtles with temperature‐dependent sex determination
Authors: Belaïd Baya , Cong Han Nguyen , Devilliers Ginette , Richard‐Mercier Noelle , Pieau Claude , Dorizzi Mireille
Published: January 2004

Cellular infiltrations forming lymphoid‐like aggregates were previously observed in gonads of two turtle species exhibiting temperature‐dependent sex determination (TSD): at hatching in Chelydra serpentina ; at and after hatching in Emys orbicularis. We show here that such aggregates are also present in gonads of Testudo graeca by the end of embryonic development, suggesting that their occurrence is general in turtles. Since in C. serpentina, infiltrations were observed mainly in testes exhibiting remnants of the germinal epithelium, it was assumed that their occurrence was an expression of maleness leading to rejection of this epithelium. The generality of this hypothesis was tested in E. orbicularis by looking for lymphoid‐like aggregates in three types of gonads (testes, ovotestes, and ovaries) and for the stages at which they occur. Gonads were from embryos, hatchlings, and young incubated at various temperatures. Ovotestes obtained by treatment with an aromatase inhibitor of eggs incubated at female‐producing temperature were also examined. In these gonads, the differentiation of Sertoli cells in testicular cords/tubes was ascertained by expression of SOX9. Moreover, the cell composition of aggregates was determined on electron micrographs. Aggregates appear in ovaries and ovotestes by the end of embryonic development and are present in the majority of these gonads at hatching, and at least up to one year after hatching. They are composed mainly of lymphocytes and fibroblasts. Aggregates are not present in typical testes. Since they occur in most ovaries, they cannot be seen as an expression of maleness. Rather, lymphocytic infiltration and formation of lymphoid aggregates in turtle gonads can be seen as components of the immune system, and can be under the control of gonadal endogenous sex steroids. J. Exp. Zool. 301A:160–168, 2004. © 2004 Wiley‐Liss, Inc.
Pluripotency of cryopreserved blastomeres of the goldfish
Authors: Kusuda Satoshi , Teranishi Tetsuo , Koide Nobuhisa , Nagai Terumi , Arai Katsutoshi , Yamaha Etsuro
Published: January 2004

To examine the pluripotency of cryopreserved blastomeres, we transplanted them into blastula. Donor blastomeres were prepared from blastula of goldfish (Carassius auratus) and cryopreserved in liquid nitrogen for two months. Fifty‐five percent and 44% of blastomeres survived after thawing. Cryopreserved blastomeres were transplanted to the blastula of triploid crucian carp (C. a. longsdorfii), which reproduces gynogenetically in nature. At four days after the operation, resultant chimeric embryos transplanted with cryopreserved blastomeres showed a survival rate (41.6%) lower than that of embryos transplanted with unfrozen blastomeres (57.1%). Transplanted blastomeres were histologically identified in various organs derived from all three germ layers. A primordial germ cell differentiated from a cryopreserved blastomere was detected in one of the 32 chimeric fish examined. These results suggest blastomeres that survive after cryopreservation retain their pluripotency and are able to differentiate into both somatic and germ cell lines. J. Exp. Zool. 301A:131–138, 2004. © 2004 Wiley‐Liss, Inc.
Aggregation effects on anhydrobiotic survival in the tardigrade Richtersius coronifer
Authors: Ivarsson Helen , JÖnsson K. Ingemar
Published: January 2004

For anhydrobiotic metazoans the rate of desiccation is an important factor influencing the probability of survival in a dry anhydrobiotic state. Formation of animal aggregations, in which the exposed body surface area of individual animals is reduced, represents one way to reduce the rate of evaporation. Such aggregations have earlier been documented in e.g., nematodes. We experimentally evaluate the effect of aggregation size (number of animals in a group of desiccating animals) on anhydrobiotic survival in the eutardigrade Richtersius coronifer. The experiment shows that aggregation provides a clear improvement on anhydrobiotic survival. The most likely explanation for this is that aggregated animals were exposed to a lower rate of desiccation. Although the empirical evidence of aggregation in tardigrades is scarce, our study suggests that aggregation could potentially be an important survival factor for tardigrades living in environments characterized by periods of rapid desiccation. J. Exp. Zool. 301A:195–199, 2004. © 2004 Wiley‐Liss, Inc.
Cyclic AMP‐dependent tyrosine phosphorylation in tammar wallaby (Macropus eugenii) spermatozoa
Authors: Bennetts Liga , Lin Minjie , Aitken R. John
Published: January 2004

Despite considerable advances in our understanding of the molecular mechanisms regulating eutherian sperm function, there is a paucity of such knowledge for the Metatheria. In eutherian spermatozoa, the attainment of functional competence is associated with a redox‐regulated, cAMP‐mediated tyrosine phosphorylation cascade, activated during capacitation. In this report we investigate whether tammar wallaby (Macropus eugenii) spermatozoa possess a similar signal transduction pathway. Western blot analysis of phosphotyrosine expression in caudal and ejaculated populations of tammar spermatozoa revealed that elevation of intracellular cAMP levels, but not exposure to oxidants or NADPH, induced a dramatic increase in the overall level of tyrosine phosphorylation. Washed, ejaculated spermatozoa exhibited more pronounced increases in tyrosine phosphorylation than unwashed sperm populations. Localisation of tyrosine phosphorylation by immunocytochemistry showed that phosphotyrosine residues were principally located along the tammar sperm flagellum, and occasionally at a small region of the sperm head, adjacent to the acrosome. Associated with the tyrosine phosphorylation of tammar spermatozoa, was a change in sperm head conformation to a T‐shaped orientation, further implying the importance of these pathways to normal tammar sperm function. Redox activity, as detected by lucigenin‐dependent chemiluminescence, was stimulated by NADPH in caudal sperm preparations but not ejaculated spermatozoa. However, neither sperm population responded to treatment with NADPH with changes in intracellular cAMP or tyrosine phosphorylation. In conclusion, tammar spermatozoa possess the same cAMP‐mediated, tyrosine phosphorylation‐dependent signal transduction cascade that has been associated with capacitation in eutherian spermatozoa. However in Metatherian spermatozoa we could find no evidence that this pathway was redox regulated. J. Exp. Zool. 301A:118–130, 2004. © 2004 Wiley‐Liss, Inc.
Ligand effects upon deuterium exchange in arenes mediated by [Ir(PR3)2(cod)]+.BF4−
Authors: Ellames George J. , Gibson Jennifer S. , Herbert John M. , Kerr William J. , McNeill Alan H.
Published: January 2004

A series of complexes of general form [Ir(PR3)2(cod)]+ has been prepared and used, without isolation, to mediate deuteration of a range of model substrates. The data suggest that, with many substrates, basicity of the phosphine ligands bound to iridium is an important factor influencing substrate selectivity and the efficiency of deuteration. In addition, the spectrum of activity of iridium complexes bearing pure donor ligands is different in many cases to that of complexes where the ligands are known to be π‐acids. Copyright © 2003 John Wiley & Sons, Ltd.
Synthesis of tritium labelled 3(R)‐HETE and 3(R),18(R/S)‐DiHETE through a common synthetic route
Authors: Groza Natalya V. , Ivanov Igor V. , Romanov Stepan G. , Shevchenko Valery P. , Myasoedov Nikolai F. , Nigam Santosh , Myagkova Galina I.
Published: January 2004

An efficient procedure for the synthesis of 3‐hydroxyoxylipins labelled with tritium on all double bond positions is reported. The synthetic scheme involves a joint route for the formation of tetraacetylenic precursors followed by stereoselective reduction of the triple bonds either with hydrogen or tritium. The final tritiated products were obtained with specific activities ranging from 1.65 to 1.80 Ci/mmol. Copyright © 2003 John Wiley & Sons, Ltd.
Synthesis of benzoxazole‐[phenyl‐13C6] by directed ortho‐metalation chemistry
Authors: Derdau Volker
Published: January 2004

A new method for the preparation of benzoxazole‐[phenyl‐13C6] (1) starting from aniline‐[13C6] (4) has been developed involving directed ortho‐metalation (DoM) chemistry. The synthesis comprises four steps and an overall yield of 39% was obtained. Copyright © 2003 John Wiley & Sons, Ltd.
Synthesis of [2H8]‐enterolactone and [2H10]‐enterodiol
Authors: Leppälä Eija , Wähälä Kristiina
Published: January 2004

Enterolactone and enterodiol are the main mammalian metabolites of dietary butyrolactone type lignans. The study of biological properties and potential health effects of these compounds requires isotopically labelled compounds as standards for quantitative measurements. An expedient deutero‐labelling method for enterolactone is to use the D3PO4·BF3/D2O complex at room temperature which will exchange all eight aromatic hydrogens, even from inactivated meta positions, to form [2,4,5,6,2′,4′,5′,6′‐2H8]‐enterolactone in 74% yield and 99% isotopic purity. [2,4,5,6,9,9,2′,4′,5′,6′‐2H10]‐Enterodiol was prepared from [2,4,5,6,2′,4′,5′,6′‐2H8]‐enterolactone by reduction with LiAlD4 which introduces two more deuterium atoms into the molecule. Copyright © 2003 John Wiley & Sons, Ltd.
Synthesis of carbon‐14 labelled l,5 diaryl‐[5‐14C]‐l,2,3‐triazolines
Authors: Matloubi Hojatollah , Shafiee Abbas , Saemian Nader , Shirvani Gholamhossein , Daha Fariba Johari
Published: January 2004

Two 1,2,3‐triazoline anticonvulsants, 1‐(4‐methylsulphone‐phenyl)‐5‐(4‐methoxy‐phenyl)‐1,2,3‐triazoline and l‐(4‐methylsulphone‐phenyl)‐5‐phenyl‐1,2,3‐triazoline, both labelled with carbon‐14 in the 5‐position, have been synthesized as part of a 5‐step sequence. Copyright © 2003 John Wiley & Sons, Ltd.
Preparation of fluorine‐18‐labelled fluoromisonidazole using two different synthesis methods
Authors: Kämäräinen Eeva‐Liisa , Kyllönen Teija , Nihtilä Outi , Björk Heikki , Solin Olof
Published: January 2004

18F‐labelled fluoromisonidazole [1H‐1‐(3‐[18F]fluoro‐2‐hydroxypropyl)‐2‐nitroimida‐zole; ([18F]FMISO)] is used as an in vivo marker of hypoxic cells in tumours and ischaemic areas of the heart and the brain. The compound plays an important role in evaluating the oxygenation status in tumours during radiotherapy. In this paper, we report experiments carried out in our laboratory in synthesizing [18F]FMISO using two different methods. The first method (I) for the [18F]FMISO synthesis was the fluorination of (2R)‐(−)‐glycidyl tosylate to [18F]epifluorohydrin. The subsequent nucleophilic ring opening, achieved with 2‐nitroimidazole, leads to labelled FMISO. The second method (II) was the fluorination of the protected precursor 1‐(2′‐nitro‐1′‐imidazolyl)‐2‐O‐tetrahydropyranyl‐3‐O‐toluenesulphonyl‐propanediol, followed by a rapid removal of the protecting group. With the first method, the radiochemical yield was about 10% at the end of the synthesis (EOS), and the radiochemical purity was over 99%. The radiochemical yield in the second method was 21% (EOS) on an average, and the radiochemical purity was over 97%. When an automated commercial synthesis module was used with method II, slightly better and more reproducible yields were achieved. The improvement in the synthesis yield with the automated apparatus will be valuable when working with high activities, and therefore it is under further development. Copyright © 2003 John Wiley & Sons, Ltd.
Quality control methods for 99mTc‐labeled exogenous natural surfactant (99mTc‐ENS)
Authors: Calmanovici Gabriela P. , Caro Ricardo A. , Goldman Cinthia G. , Leonardi Natalia M. , Boccio José R. , Zubillaga Marcela B.
Published: January 2004

To standardize the quality control for 99mTc‐ENS, the following methods were studied: (1) physical properties and pH, (2) radiochemical purity (chromatographic studies on Whatman‐1 paper, or instant thin‐layer chromatography and solvent extraction using different solvents and (3) rat biodistribution studies by intratracheal injection. The tolerance limits were fixed for each method. The radiopharmaceutical stability was also evaluated. The results showed that 99mTc‐ENS was a white suspension with a pH between 4.0 and 6.0. The limit for radiochemical impurities in Whatman‐1 paper/acetone was fixed at lower than 2% and the established limit for the organic aliquot in cyclohexane extraction was greater than 2%. In the biodistribution studies, the limits for activity concentration were fixed at greater than 90% for lungs, less than 9% for the gastrointestinal system and less than 1% for the sum of the other organs studied. After a storage time of 6 h at room temperature or in a refrigerator, 99mTc‐ENS physical properties and pH, radiochemical and biodistribution results were within the established values. In conclusion, the quality control methods for 99mTc‐ENS are tests on physical properties and pH, radiochemical purity by Whatman‐1 paper/acetone chromatography and cyclohexane extraction and biodistribution studies in rats. The stability of this radiopharmaceutical is at least 6 h at room temperature. Copyright © 2003 John Wiley & Sons, Ltd.
Flixotide™‐pressurized metered‐dose inhalers loaded with [18F]fluticasone propionate particles for drug deposition studies in humans with PET–formulation and analysis
Authors: Constantinou Maria , Waters Stephen L. , Steel Colin J. , Poole Keith G. , Marino Phil S. , Ind Phillip W. , Rhodes Christopher G. , Aigbirhio Franklin I. , Moore Alison , Pike Victor W. , Luthra Sajinder K.
Published: January 2004

Fluticasone propionate (FP) is a potent anti‐inflammatory synthetic steroid, used for the treatment of asthma. Flixotide™ is a formulated pressurized metered‐dose inhaler (pMDI) that contains small‐micronized FP particles in a blend of CFC propellants. Our objective was to develop a radiotracer method for accurately measuring the regional deposition of FP within the human lung using positron emission tomography (PET), which would be of important clinical interest. Flixotide™ pMDIs were used to prepare [18F]FP pMDIs labeled isotopically with the positron emitter, fluorine‐18 (t1/2=109.7 min). FP particles from Flixotide™ pMDIs were mixed with [18F]FP formulated into a pMDI and sonicated at room temperature. The drug delivery of [18F]FP pMDI (250 μg of FP per actuation dose) was assessed for particle size distribution and dose uniformity. The distributions of FP and [18F]FP across particle size in such preparations were measured with an Andersen cascade impactor. This procedure was shown to provide an emitted dose from a [18F]FP pMDI of 246±19 μg/per metered dose. The particle size distribution as measured by mass median aerodynamic diameter (MMAD) (The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) for each distribution were calculated. MMAD is defined as the aerodynamic diameter around which the mass of particles is equally distributed and the GSD is a measure of the dispersion of these particle diameters around the MMAD) from a commercial Flixotide™ pMDI was 2.6±0.2 μm and agreed well with that from an [18F]FP pMDI (2.8±0.1 μm). The MMAD and geometric standard deviation (GSD) of newly formulated [18F]FP pMDIs were unaffected by the formulation procedure. [18F]FP was distributed with good uniformity with respect to the mass of FP for particles greater than 0.43 μm. Hence, the radiolabeled pMDI is a suitable source of radiotracer for the regional measurement of lung deposition for inhaled FP in human subjects with PET. Copyright © 2003 John Wiley & Sons, Ltd.
A 11C‐Methyl stannane (5‐[11C]methyl‐1‐aza‐5‐stanna‐bicyclo[3.3.3]undecane) for use in palladium‐mediated [11C]C–C bond forming reactions with organohalides
Authors: Forngren Tobias , Samuelsson Linda , Långström Bengt
Published: January 2004

The synthesis of a 11C‐labelled methyl stannane, (5‐[11C]methyl‐1‐aza‐5‐stanna‐bicyclo[3.3.3]undecane (2)), and its use in palladium‐mediated Stille reactions to form [11C]C–C bonds are described. Stannane 2 was synthesized from iodo[11C]methane, 5‐chloro‐1‐aza‐5‐stanna‐bicyclo[3.3.3]undecane 1 and butyl lithium in 20–90% decay‐corrected radiochemical yield starting from iodo[11C]methane. Subsequent reaction with a series of substituted aryl and vinyl halides produced the corresponding [11C]methylated products 3–5 in up to 90% decay‐corrected radiochemical yield from the crude 2. The total synthesis time, including purification, was 25–30 min from end of radionuclide production. Copyright © 2003 John Wiley & Sons, Ltd.
Microwave accelerated 68Ga‐labelling of oligonucleotides
Authors: Velikyan I. , Lendvai G. , Välilä M. , Roivainen A. , Yngve U. , Bergström M. , Långström B.
Published: January 2004

Oligonucleotides are extensively used for characterization of gene expression in vitro and have now been studied as inhibitors of gene expression in vivo in various diseases. Labelled antisense oligonucleotides are therefore of potential interest for possible in vivo imaging of gene expression, considering the biology of tumors and applications in designing novel molecule‐targeted therapies. In the present work a method of microwave accelerated 68Ga‐labelling of oligonucleotides and analysis of the resulting tracers are described.

Four modified and functionalized 17‐mer oligonucleotides with a hexylamine group in the 3′‐ or 5′‐position were studied. The oligonucleotides were conjugated to the bifunctional chelator, 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA), and then labelled with 68Ga(T1/2=68 min) using microwave activation. The isolated decay‐corrected radiochemical yields ranged from 30 to 52%. Labelled products were stable in water and ethanol for more than 4 h. The impact of the labelling procedure on the oligonucleotide probes was investigated using hybridization to a complementary 17‐mer sense oligonucleotide in solution. Chemical modification did not influence either the labelling or hybridization ability of the oligonucleotides. The radiolabelled oligonucleotides will be used for the further in vitro and in vivo biology studies. Copyright © 2003 John Wiley & Sons, Ltd.
An asymmetric synthesis of l‐[3‐13C]alanine
Authors: Takatori Kazuhiko , Toyama Sanae , Narumi Shoko , Fujii Seishiro , Kajiwara Masahiro
Published: January 2004

l‐[3‐13C]Alanine was synthesized from [13C]methyl iodide by using Dellaria's oxazinone, prepared from phenyl[2‐13C]bromoacetate and (S)‐2‐phenylglycinol, as a chiral glycine equivalent. Alkylation of the oxazinone with [13C]methyl iodide was achieved with high diastereoselectivity. Hydrolysis and removal of the chiral auxiliary of the alkylated oxazinone gave l‐[3‐13C]alanine. Copyright © 2003 John Wiley & Sons, Ltd.
Wall shear rates differ between the normal carotid, femoral, and brachial arteries: An in vivo MRI study
Authors: Wu Sheng Ping , Ringgaard Steffen , Oyre Sten , Hansen Michael S. , Rasmus Stokholm , Pedersen Erik M.
Published: January 2004

Purpose

To investigate wall shear rates in vivo in the common carotid, brachial, and superficial femoral arteries using very high resolution magnetic resonance imaging (MRI) phase contrast measurements.

Materials and Methods

Mean, maximum, and minimum wall shear rate and an oscillatory shear index were measured for 20 volunteers, aged 23.3 ± 1.9 years, in the three arteries, using phase contrast imaging with 0.0625 mm2 resolution and three‐dimensional paraboloid fitting.

Results

The superficial femoral artery had the lowest mean (130.3 ± 13.1 second−1), maximum (735.8 ± 32.4 second−1), and minimum (–224.5 ± 17.0 second−1) wall shear rate, as well as the highest oscillatory shear index (0.21 ± 0.02). All values were significantly different (P < 0.05) from both the brachial artery and the common carotid artery values. The highest mean (333.3 ± 13.6 second−1) and minimum (117.9 ± 24.5 second−1) wall shear rates and the lowest oscillatory shear index (0 ± 0) were found in the common carotid artery.

Conclusion

It is possible to measure wall shear rate in vivo in different arteries using MRI with very high resolution. The findings exhibit the in vivo environment of wall shear rates and suggest a nonuniform distribution of wall shear rates throughout the arterial system. J. Magn. Reson. Imaging 2004;19:188–193. © 2004 Wiley‐Liss, Inc.
Is half‐dose contrast‐enhanced three‐dimensional MR angiography sufficient for the abdominal aorta and pelvis?
Authors: Takahashi Satoru , Murakami Takamichi , Takamura Manabu , Kim Tonsok , Hori Masatoshi , Narumi Yoshifumi , Nakamura Hironobu
Published: January 2004

Purpose

To evaluate the usefulness of half‐dose contrast‐enhanced magnetic resonance (MR) angiography for depicting the abdominal aorta and its major branches.

Materials and Methods

A total of 72 consecutive patients were randomly assigned to one of four groups that underwent MR angiography after receiving different concentrations (original or diluted to 50%) and total amounts (single or half‐dose) of gadolinium chelate injected at different rates (1 or 0.5 mL/second). The signal‐to‐noise ratio (SNR) and contrast‐to‐noise ratio (CNR) of the abdominal aorta and of the common and external iliac arteries were calculated, and two blinded readers rated the respective image qualities.

Results

The SNR and CNR of the abdominal aorta and the common iliac artery in the 0.5 mL/second groups were statistically significantly lower than those in the 1 mL/second groups. The differences in overall image quality across the four groups were not statistically significant.

Conclusion

Half‐dose MR angiography using diluted contrast medium injected at a rate of 1 mL/second depicted the abdominal aorta and its branches as clearly as using a full single dose. J. Magn. Reson. Imaging 2004;19:194–201. © 2004 Wiley‐Liss, Inc.