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Cell clarification represents a major challenge for the intensification through very high cell density in the production of biopharmaceuticals such as monoclonal antibodies (mAbs). The present report proposes a solution to this challenge in a streamlined process where cell clarification and mAb capture are performed in a single step using magnetic beads coupled with protein A. Capture of mAb from non-clarified CHO cell suspension showed promising results; however, it has not been demonstrated that it can handle the challenge of very high cell density as observed in intensified fed-batch cultures. The performances of magnetic bead-based mAb capture on non-clarified cell suspension from intensified fed-batch culture were studied. Capture from a culture at density larger than 100 × 106 cells/ml provided an adsorption efficiency of 99% and an overall yield of 93% with a logarithmic host cell protein (HCP) clearance of ≈2–3 and a resulting HCP concentration ≤≈5 ppm. These results show that direct capture from very high cell density cell suspension is possible without prior processing. This technology, which brings significant benefits in terms of operational cost reduction and performance improvements such as low HCP, can be a powerful tool alleviating the challenge of process intensification. QC 20221102

A simple method was employed for the synthesis of dihydropyrano[3, 2-b]chromenedione derivatives (4a-o) in high yields by condensation of 5, 5-dimethylcyclohexane-1, 3-dione(1), different aromatic aldehydes (2a-o), and 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one(3), using meglumine as a stable and reusable catalyst. Meglumine, an amino sugar, was employed as an environmentally benign catalyst, due to its splendid properties such as being inexpensive, recyclable, and biodegradable. The accomplished protocol employs low catalyst loading and easy work-up for the synthesis of 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one derivatives. A great asset is that without any significant loss, the catalyst could be recovered and reused for extended synthetic steps. This offer huge advantage to overcome recyclability issues. Our synthesized compounds were analyzed by IR, 1H, 13C NMR, mass spectra and evaluated for their antioxidant properties by 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH), hydrogen peroxide(H2O2), and nitric oxide (NO) scavenging methods. The correlation in exhibition of antioxidant activity was effective at all doses. The binding interactions and molecular docking studies for entitled compounds were studied against 3MNG protein; 4k exhibited marked binding affinity with excellent docking score of −7.6 Kcal/mol and emerged as a lead compound. © 2019 Wiley Periodicals, Inc. Ural Federal University, UrFU The authors G. Sravya and N. Bakthavatchala Reddy are thankful to Ural Federal University, Yekaterinburg, Russia, for postdoctoral fellowship.

Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the vessel wall. Macrophages depend on their polarization to exert either pro-inflammatory or anti-inflammatory effects. Macrophages of the anti-inflammatory phenotype express high levels of CD163, a scavenger receptor for the hemoglobin-haptoglobin complex. CD163 can also bind to the pro-inflammatory cytokine TWEAK. Using ApoE-deficient or ApoE/CD163 double-deficient mice we aim to investigate the involvement of CD163 in atherosclerosis development and its capacity to neutralize the TWEAK actions. ApoE/CD163 double-deficient mice displayed a more unstable plaque phenotype characterized by an increased lipid and macrophage content, plaque size, and pro-inflammatory cytokine expression. In vitro experiments demonstrated that the absence of CD163 in M2-type macrophages-induced foam cell formation through upregulation of CD36 expression. Moreover, exogenous TWEAK administration increased atherosclerotic lesion size, lipids, and macrophages content in ApoE-/-/CD163-/- compared with ApoE-/-/CD163+/+ mice. Treatment with recombinant CD163 was able to neutralize the proatherogenic effects of TWEAK in ApoE/CD163 double-deficient mice. Recombinant CD163 abolished the pro-inflammatory actions of TWEAK on vascular smooth muscle cells, decreasing NF-kB activation, cytokines and metalloproteinases expression, and macrophages migration. In conclusion, CD163-expressing macrophages serve as a protective mechanism to prevent the deleterious effects of TWEAK on atherosclerotic plaque development and progression. © 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology

Spermatogonial stem cell (SSC) is known for its self-renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC-DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA-. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA- were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA- in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA- in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA-. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA- (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC.

Neurocysticercosis is a parasitic brain disease caused by the larval form (Cysticercus cellulosae) of the Taenia solium and is the leading cause of preventable epilepsy world-wide. However, the pathophysiology and relation to the wide range of clinical features remains poorly understood. Axonal swelling is emerging as an important early pathological finding in multiple neurodegenerative diseases and causes of brain injury, but has not been well-described in neurocysticercosis. Histological analysis was performed on human, rat and porcine NCC brain specimens to identify axonal pathology. Rat infection was successfully carried out via two routes of inoculation: direct intracranial injection and oral feeding. Extensive axonal swellings, in the form of spheroids, were observed in both humans and rats and to a lesser extent in pigs with NCC. Spheroids demonstrated increased immunoreactivity to amyloid precursor protein and neurofilament indicating probable impairment of axonal transport. These novel findings demonstrate that spheroids are present in NCC which is conserved across species. Not only is this an important contribution towards understanding that pathogenesis of NCC, but it also provides a model to analyze the association of spheroids with specific clinical features and to investigate the reversibility of spheroid formation with antihelmintic treatment.

Lysyl oxidase (LOX) controls matrix remodeling, a key process that underlies cardiovascular diseases and heart failure; however, a lack of suitable animal models has limited our knowledge with regard to the contribution of LOX to cardiac dysfunction. Here, we assessed the impact of LOX overexpression on ventricular function and cardiac hypertrophy in a transgenic LOX (TgLOX) mouse model with a strong cardiac expression of human LOX. TgLOX mice exhibited high expression of the transgene in cardiomyocytes and cardiofibroblasts, which are associated with enhanced LOX activity and H2O2 production and with cardiofibroblast reprogramming. LOX overexpression promoted an age-associated concentric remodeling of the left ventricle and impaired diastolic function. Furthermore, LOX transgenesis aggravated angiotensin II (Ang II)–induced cardiac hypertrophy and dysfunction, which triggered a greater fibrotic response that was characterized by stronger collagen deposition and cross-linking and high expression of fibrotic markers. In addition, LOX transgenesis increased the Ang II–induced myocardial inflammatory infiltrate, exacerbated expression of proinflammatory markers, and decreased that of cardioprotective factors. Mechanistically, LOX overexpression enhanced oxidative stress and potentiated the Ang II–mediated cardiac activation of p38 MAPK while reducing AMPK activation. Our findings suggest that LOX induces an age-dependent disturbance of diastolic function and aggravates Ang II–induced hypertrophy, which provides novel insights into the role of LOX in cardiac performance. © FASEB.

Implanted polymeric materials, such as medical devices, provoke the body to initiate an inflammatory reaction, known as the foreign body response (FBR), which causes several complications. In this study, polyurethane (Tecoflex�, PU) surface modified with the nonionic surfactant Tween80� (PU/T80) and the cell adhesive PLL-RGD peptide (PU/PLL-RGD) by a previously described entrapment technique were implanted in the peritoneal cavity of Wistar rats for 30 days. Implants were retrieved and examined for tissue reactivity and cellular adherence by various microscopic and analytical techniques. Surface-induced inflammatory response was assessed by real-time PCR based quantification of proinflammatory cytokine transcripts, namely, TNF-? and IL-1?, normalized to housekeeping gene GAPDH. Cellular adherence and their distribution profile were assessed by microscopic examination of H&E stained implant sections. It was observed that PU/PLL-RGD followed by the bare PU surface exhibited severe inflammatory and fibrotic response with an average mean thickness of 19 and 12 I?m, respectively, in 30 days. In contrast, PU/T80 surface showed only a cellular monolayer of 2-3 I?m in thickness, with a mild inflammatory response and no fibrotic encapsulation. The PU/PLL-RGD peptide-modified substrate promoted an enhanced rate of macrophage cell fusion to form foreign body giant cell (FBGCs), whereas FBGCs were rarely observed on Tween80�-modified substrate. The expression levels of proinflammatory cytokines (TNF-? and IL-1?) were upregulated on PU/PLL-RGD surface followed by bare PU, whereas the cytokine expressions were significantly suppressed on PU/T80 surface. Thus, our study highlights modulation of foreign body response on polyurethane surfaces through surface entrapment technique in the form of differential responses observed on PLL-RGD and Tween80� modified surfaces with the former effective in triggering tissue cell adhesion thereby fibrous encapsulation, while the later being mostly resistant to this phenomenon. � 2010 Wiley Periodicals, Inc.

Collagen was isolated and purified from sheep intestines and cast into a film. The collagen film was cross-linked with ultraviolet light and reinforced by imbedding a nylon mesh. The collagen membrane was evaluated as a wound dressing for second- and third-degree burns in rats. Histopathological studies of the burns with and without the collagen dressings were made. The study showed the burn wounds dressed with collagen membrane exhibited rapid epithelialization with little inflammatory change and dense fibrous tissue ingrowth in the subdermal layers.

Proper cell attachment and distribution, and thus stronger association in vivo between a bone implant and native tissue will improve the success of the implant. In this study, the aim was to achieve promotion of attachment and uniform distribution of rat mesenchymal stem cell-derived osteoblasts by introducing chemical and topographical cues on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film surfaces. As the chemical cues, either alkaline phosphatase was covalently immobilized on the film surface to induce deposition of calcium phosphate minerals or fibrinogen was adsorbed to improve cell adhesion. Microgrooves and micropits were introduced on the film surface by negative replication of micropatterned Si wafers. Both chemical cues improved cell attachment and even distribution on the PHBV films, but Fb was more effective especially when combined with the micropatterns. Cell alignment (

Conversion of the cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is the hallmark of prion diseases, which are fatal and transmissible neurodegenerative disorders. ER-retained anti-prion recombinant single-chain Fv fragments have been proved to be an effective tool for inhibition of PrP(C) trafficking to the cell surface and antagonize PrP(Sc) formation and infectivity. In the present study, we have generated the secreted version of 8H4 intrabody (Sec-8H4) in order to compel PrP(C) outside the cells. The stable expression of the Sec-8H4 intrabodies induces proteasome degradation of endogenous prion protein but does not influence its glycosylation profile and maturation. Moreover, we found a dramatic diverting of PrP(C) traffic from its vesicular secretion and, most importantly, a total inhibition of PrP(Sc) accumulation in NGF-differentiated Sec-8H4 PC12 cells. These results confirm that perturbing the intracellular traffic of endogenous PrP(C) is an effective strategy to inhibit PrP(Sc) accumulation and provide convincing evidences for application of intracellular antibodies in prion diseases.

The antimitotic agent paclitaxel continues to play an important role in cancer chemotherapy. However, its inefficacy on certain resistant cells and toxic side effects had led to the search for new drugs with improved biological activity. Here the QSAR models for microtubule stabilizing anticancer agents were performed to correlate their physicochemical properties with biological activity. Single and multiple linear regression models for six cancer cell lines were obtained with R2 ? 0.65 and q2pre ? 0.6. Molecular mechanics energy and log P of the molecules account for the activity of taxanes towards B16 melanoma and breast cancer cells, respectively. The lowest unoccupied molecular orbitals and the number of nitrogen atoms in the structure account for the biological activity of epothilone derivatives and rest of the drugs towards ovarian cells. The relation between the structural properties of microtubule stabilizing antimitotic compounds and their activities on different cell lines are investigated in this paper. � 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Islet amyloid deposition is increasingly seen as a pathogenic feature of type 2 diabetes mellitus (T2DM), with the deposits containing the unique amyloidogenic peptide islet amyloid polypeptide (IAPP, also known as amylin). The fibril precursors of IAPP contribute to its cytotoxicity on pancreatic β cells and be important in causing β-cell dysfunction in T2DM. However, the development of effective this study, inhibitors against the toxicity of IAPP has been extremely challenging. We have found that pre-incubation with geniposide dose-dependently prevented human IAPP (hIAPP)-induced cell damage in INS-1E cells, and bacitracin, an inhibitor of IDE activity, prevented significantly the protective effects of geniposide in pancreatic INS-1E cells significantly. Geniposide induced the expression of insulin-degrading enzyme (IDE), a key degrading protein of hIAPP, but had no significant effect on the aggregation of hIAPP. These findings indicate that geniposide prevents hIAPP-induced cytotoxicity in INS-1E cells involving upregulation of IDE expression.

Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt-5a, Wnt-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1–10), growth (days 5–12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt-7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies. © 2004 Wiley-Liss, Inc.

Experimental epileptic models have been developed to assess the pathophysiology of epileptic seizures. We have previously shown that epileptic seizures cause significant neuronal loss in hippocampus (p0.05). This study suggests that the loss of hippocampal neurons in penicillin-induced epilepsy seizure is not directly correlated with a volume change in the hippocampus. Furthermore, our results indicate that it is important to assess both the neuron number and the volume of the affected area using stereological methods in an epilepsy model to objectively analyze the effects of the seizure.

Ascidia malaca gametes before fertilization incubated in 10-5 or 10-7 M solutions of tributyltin(IV) chloride, TBTCl, for 3 h appear highly damaged under transmission electron microscopy observation. Also, the fertilization process is affected by the compound: the damaged spermatozoa are present in the vitelline coat and the egg does not cleave. An increase of microbodies, structurally similar to peroxisomes, have been detected in the egg peripheral cytoplasm, probably in relation to their role in alleviating damage to some cellular components. The results have shown that the reproduction of ascidians under unfavourable environmental conditions is prevented. Copyright © 2003 John Wiley & Sons, Ltd..

The realization that the mammalian proteomic complexity is achieved with a limited number of genes demands a better understanding of alternative splicing regulation. Promoter control of alternative splicing was originally described by our group in studies performed on the fibronectin gene. Recently, other labs extended our findings to the cystic fibrosis, CD44 and CGRP genes strongly supporting a coupling between transcription and pre-mRNA splicing. A possible mechanism that would fit in these results is that the promoter itself is responsible for recruiting splicing factors, such as SR proteins, to the site of transcription, possibly through transcription factors that bind the promoter or the transcriptional enhancers. An alternative model, discussed more extensively in this review, involves modulation of RNA pol II (pol II) elongation rate. The model is supported by findings that cis- and trans-acting factors that modulate pol II elongation on a particular template also provoke changes in the alternative splicing balance of the encoded mRNAs. Fil:Nogués, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kadener, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Cramer, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:De la Mata, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Fededa, J.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Blaustein, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Srebrow, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kornblihtt, A.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Objectives: Successful cancer treatments still require more compounds to be isolated from natural sources. Thus, we have investigated anti-proliferative/apoptotic effects of methanolic extracts of lichen species Parmelia sulcata Taylor and Usnea filipendula Stirt on human lung cancer (A549, PC3), liver cancer (Hep3B) and rat glioma (C6) cells. Materials and methods: Anti-proliferative effects were monitored by MTT and adenosine triphosphate viability assays, while genotoxic activity was studied using the comet assay. Additionally, cell death mode and apoptosis assays (fluorescence staining, caspase-cleaved cytokeratin 18, caspase-3 activity and PARP cleavage) were performed. Results: Extracts produced anti-population growth effects in a dose-dependent manner (1.56–100 lg/ ml) by inducing apoptosis-like cell death. This resulted in the lines having the presence of pyknotic cell nuclei. In addition, significant increase in genetic damage in the cell lines was seen, indicating that DNA damage may have been responsible for apoptotic cell death. Conclusion: In this study, methanolic extracts of Parmelia sulcata and Usnea filipendula induced apoptosis-like cell death by causing DNA damage, to cancer cells.

We have studied the patterns of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) co-expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet-inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4 and IFN-gamma were determined by three-colour flow cytometric analysis of fixed and saponin-permeabilized cells fluorescent-stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL-2/IL-4, IL-2/IFN-gamma and IL-4/IFN-gamma. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus-immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co-expressing combinations of them (i.e. IL-4/IL-2, IL-4/IFN-gamma and IL-2/IFN-gamma), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.

Lysosomes concentrate juxtanuclearly in the region around the microtubule-organizing center by interaction with microtubules. Different experimental and physiological conditions can induce these organelles to move to the cell periphery by a mechanism implying a plus-end-directed microtubule-motor protein (a kinesin-like motor). The responsible kinesin-superfamily protein, however, is unknown. We have identified a new mouse isoform of the kinesin superfamily, KIF2β, an alternatively spliced isoform of the known, neuronal kinesin, KIF2. Developmental expression pattern and cell-type analysis in vivo and in vitro reveal that KIF2β is abundant at early developmental stages of the hippocampus but is then downregulated in differentiated neuronal cells, and it is mainly or uniquely expressed in non-neuronal cells while KIF2 remains exclusively neuronal. Electron microscopy of mouse fibroblasts and immunofluorescence of KIF2β-transiently-transfected fibroblasts show KIF2 and KIF2β primarily associated with lysosomes, and this association can be disrupted by detergent treatment. In KIF2β-overexpressing cells, lysosomes (labeled with anti-lysosome-associated membrane protein-1) become abnormally large and peripherally located at some distance from their usual perinuclear positions. Overexpression of KIF2 or KIF2β does not change the size or distribution of early, late and recycling endosomes nor does overexpression of different kinesin superfamily proteins result in changes in lysosome size or positioning. These results implicate KIF2β as a motor responsible for the peripheral translocation of lysosomes. 17 5855 5867 Cited By :50

Retinoid X receptor-gamma (RXRgamma) is a transcription factor that mediates retinoid signalling and is expressed in rat heart during adult life. However, its expression in embryonic and neonatal heart has not been investigated and it is not known whether ventricular cardiomyocytes express RXRgamma or whether all- trans -retinoic acid (tRA) and thyroid hormone (T3) could influence RXRgamma transcript levels in these cells. First, in situ hybridization experiments were used to test for any spatio-temporal correlation between RXRgamma gene expression and the previously shown requirement for retinoid signalling in embryonic ventricular cardiomyocytes. It was shown that RXRgamma transcripts are not detectable in embryonic heart at all developmental stages examined under conditions where they are detectable in other embryonic tissues. Second, Northern blotting was used to examine whether there is a difference in RXRgamma transcript levels between neonatal and adult heart. We show that levels of two RXRgamma transcripts are developmentally regulated during the postnatal period because they differ between neonatal and adult hearts. Third, it was demonstrated that primary cultures of neonatal rat ventricular cardiomyocytes express RXRgamma transcripts, making them a novel in vitro system for the study of RXRgamma gene regulation in heart-derived cells. Finally, this system was used to examine whether tRA and T3can influence levels of RXRgamma transcripts because they have been shown to have antagonistic effects in this system and to influence RXRgamma RNA levels in other systems. It was shown by Northern blot experiments, that in this system, RXRgamma transcript levels are differentially influenced by these two hormones. The significance of these findings in relation to previously published work is discussed.

The tumor microenvironment plays a pivotal role during cancer development and progression. The balance between suppressive and cytotoxic responses of the tumor immune microenvironment has been shown to have a direct effect on the final outcome in various human and experimental tumors. Recently, we demonstrated that the oxygen sensor HIF-prolyl hydroxylase-2 (PHD2) plays a detrimental role in tumor cells, stimulating systemic growth and metastasis in mice. In our current study, we show that the conditional ablation of PHD2 in the hematopoietic system also leads to reduced tumor volume, intriguingly generated by an imbalance between enhanced cell death and improved proliferation of tumor cells. This effect seems to rely on the overall downregulation of protumoral as well as antitumoral cytokines. Using different genetic approaches, we were able to confine this complex phenotype to the crosstalk of PHD2-deficient myeloid cells and T-lymphocytes. Taken together, our findings reveal a multifaceted role for PHD2 in several hematopoietic lineages during tumor development and might have important implications for the development of tumor therapies in the future. What's new? Here the authors describe the complex role of the oxygen sensor PHD2 in tumor-associated immune cells in a conditional PHD2-deficient mouse line. They demonstrate that the observed reduced tumor volume is a consequence of opposing changes including the drastic down-regulation of numerous pro- as well as anti-tumoral genes and an imbalance between enhanced cell death and greater proliferation of tumor cells. They confined this complex phenotype to the crosstalk of PHD2-deficient myeloid cells and T-lymphocytes. The findings reveal a multifaceted role for PHD2 in hematopoietic lineages during tumor development and might have important implications for the development of tumor therapies.

The interaction of bovine pancreatic trypsin inhibitor (BPTI) and bovine tryptase, which are co-localized in the same granules of bovine mast cells, has been analyzed at 30°C in 0.1 M Tris-HCl, pH 8.0. The analysis has unravelled that the functional unit of bovine tryptase is formed of (at least) four binding sites for this inhibitor. These interaction sites display a simple binding behaviour for small inhibitors (and substrates), whereas heterogeneous properties have been observed in the binding of BPTI. Furthermore, in the presence of BPTI, a positive functional interaction can be detected among the binding sites also for a small synthetic inhibitor, like benzamidine. Such features indicate the existence of a complex functional interplay among the sites of the functional unit which is transmitted through the secondary specificity sites.

Vitrification is becoming a preferred method for pre-implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo- and in vitro-produced bovine embryos after vitrification. In vitro- (IVF) and in vivo-derived (IVV) bovine blastocysts were identified as follows: in vitro-produced fresh (IVF-F), in vitro-produced vitrified (IVF-V), in vivo-derived fresh (IVV-F), in vivo-derived vitrified (IVV-V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF-F × IVV-F, IVF-V × IVV-V, IVF-F × IVF-V, and IVV-F × IVV-V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up-regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up-regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo-produced blastocysts. After vitrification, however, in vitro-produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro-produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up-regulation of genes that are involved in stress responses.

Erythropoietin acts by binding to its cell surface receptor on erythroid progenitor cells to stimulate erythrocyte production. Erythropoietin receptor expression in nonhematopoietic tissue, including skeletal muscle progenitor cells, raises the possibility of a role for erythropoietin beyond erythropoiesis. Mice with erythropoietin receptor restricted to hematopoietic tissue were used to assess contributions of endogenous erythropoietin to promote skeletal myoblast proliferation and survival and wound healing in a mouse model of cardiotoxin induced muscle injury. Compared with wild-type controls, these mice had fewer skeletal muscle Pax-7+ satellite cells and myoblasts that do not proliferate in culture, were more susceptible to skeletal muscle injury and reduced maximum load tolerated by isolated muscle. In contrast, mice with chronic elevated circulating erythropoietin had more Pax-7+ satellite cells and myoblasts with increased proliferation and survival in culture, decreased muscle injury, and accelerated recovery of maximum load tolerated by isolated muscle. Skeletal muscle myoblasts also produced endogenous erythropoietin that increased at low O2. Erythropoietin promoted proliferation, survival, and wound recovery in myoblasts via the phosphoinositide 3-kinase/AKT pathway. Therefore, endogenous and exogenous erythropoietin contribute to increasing satellite cell number following muscle injury, improve myoblast proliferation and survival, and promote repair and regeneration in this mouse induced muscle injury model independent of its effect on erythrocyte production.—Jia, Y., Suzuki, N., Yamamoto, M., Gassmann, M., Noguchi, C. T. Endogenous erythropoietin signaling facilitates skeletal muscle repair and recovery following pharmacologically induced damage.

This paper investigates the distribution of four classes of neuropeptides, myomodulin, small cardioactive peptide (SCP), buccalin, and FMRFamide, in central neurons forming the network that underlies feeding behavior in the snail Lymnaea stagnalis. Intracellular dye‐marking and immunocytochemical analysis, using antisera to the different classes of peptides, were applied to identified neurons of all three levels of the hierarchy of the circuitry: modulatory interneurons (cerebral giant cells, CGC slow oscillator, SO), central pattern generator (CPG) interneurons (N1, N2, N3), motoneurons (B1–B10), and their peripheral target organs. Myomodulin immunoreactivity was detected in the CGC interneurons, in the SO, and in ventral N2‐type CPG interneurons. Several large buccal motoneurons, the paired B1, B2, B3, B7, and neurons located in the dorsal posterior area (putative B4 cluster types) were also myomodulin immunoreactive. Target organs of buccal motoneurons, the buccal mass, salivary glands, and oesophagus contained myomodulin‐immunopositive fibers. SCP appeared in N2‐type interneurons and was found colocalized with myomodulin in the B1 and B2 motoneurons. SCP‐containing neurons in the B4 cluster area were also detected. The buccal mass and salivary glands exhibited SCP‐immunoreactive fibers. Buccalin immunoreactivity was scarce in the buccal ganglia and was identified only in N1‐type interneurons and three pairs of dorsal posterior neurons. In the periphery, immunoreactive fibers were localized in the oesophagus only. None of the buccal neuronal types examined revealed immunoreactivity to SEQPDVDDYLRDVVLQSEEPLY (“SEEPLY”), a peptide encoded in the FMRFamide precursor protein of Lymnaea. SEEPLY immunoreactivity was confined to a pair of novel ventral neurons with projections to the laterobuccal nerve innervating the buccal mass. Immunoreactive fibers were also traced in this organ. © 1994 Wiley‐Liss, Inc. Copyright © 1994 Wiley‐Liss, Inc. 342 352 365 Cited By :39

FKBP51 and FKBP52 (FK506-binding protein 51 and 52) are tetratricopeptide repeat-domain immunophilins belonging to the tetratricopeptide-protein•hsp90•hsp70•p23 heterocomplex bound to steroid receptors. Immunophilins are related to receptor folding, subcellular localization, and hormone-dependent transcription. Also, they bind the immunosuppressant macrolide FK506, which shows neuroregenerative and neuroprotective actions by a still unknown mechanism. In this study, we demonstrate that in both, undifferentiated neuroblastoma cells and embryonic hippocampal neurons, the FKBP52•hsp90•p23 heterocomplex concentrates in a perinuclear structure. Upon cell stimulation with FK506, this structure disassembles and this perinuclear area becomes transcriptionally active. The acquisition of a neuronal phenotype is accompanied by increased expression of βIII-tubulin, Map-2, Tau-1, but also hsp90, hsp70, p23, and FKBP52. During the early differentiation steps, the perinuclear heterocomplex redistributes along the cytoplasm and nascent neurites, p23 binds to intermediate filaments and microtubules acquired higher filamentary organization. While FKBP52 moves towards neurites and concentrates in arborization bodies and terminal axons, FKBP51, whose expression remains constant, replaces FKBP52 in the perinuclear structure. Importantly, neurite outgrowth is favored by FKBP52 over-expression or FKBP51 knock-down, and is impaired by FKBP52 knock-down or FKBP51 over-expression, indicating that the balance between these FK506-binding proteins plays a key role during the early mechanism of neuronal differentiation. Fil: Quintá, Héctor Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina Fil: Maschi, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina Fil: Gomez Sanchez, Celso. University of Mississippi; Estados Unidos. G. V. (Sonny) Montgomery VA Medical Center; Estados Unidos Fil: Piwien Pilipuk, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina Fil: Galigniana, Mario Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina

Several chemical modifications in the N 1-benzenesulfonamide ring of celecoxib are presented. The series with a hydroxymethyl group adjacent to the sulfonamide was found to be the most potent modification that yielded many compounds selectively active against COX-2 enzyme in vitro. ? 2003 Elsevier Ltd. All rights reserved.