bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatase 2A is involved in endothelial cell microtubule remodeling and barrier regulation
Authors: Tar Krisztina , Birukova Anna A. , Csortos Csilla , Bakó Éva , Garcia Joe G.N. , Verin Alexander D.
Published: May 2004

We have recently shown that microtubule (MT) inhibitor, nocodazole (2–5 μM) significantly increases endothelial cells (EC) actomyosin contraction and permeability indicating the importance of MT in maintaining the EC barrier (Verin et al. [2001]: Cell Mol Physiol 281:L565–L574). Okadaic acid (OA, 2–5 nM), a powerful inhibitor of protein phosphatase 2A (PP2A), significantly potentiates the effect of submaximal concentrations of nocodazole (50–200 nM) on transendothelial electrical resistance (TER) suggesting the involvement of PP2A activity in the MT‐mediated EC barrier regulation. Immunofluorescent staining of EC revealed that in control cells PP2A distributes in a pattern similar to MT. Consistent with these results, we demonstrated that significant amounts of PP2A were present in MT‐enriched EC fractions indicating tight association of PP2A with MT in endothelium. Treatment of EC with OA leads to disappearance of MT‐like PP2A staining suggesting dissociation of PP2A from the MT network. Next, we examined the effect of PP2A inhibition on phosphorylation status of MT‐associated protein tau, which in its unphosphorylated form promotes MT assembly. OA caused significant increases in tau phosphorylation confirming that tau is a substrate for PP2A in endothelium. Immunofluorescent experiments demonstrated that the OA‐induced increases in tau phosphorylation strongly correlated with translocation of phospho‐tau to cell periphery and disassembly of peripheral MT. These results suggest the involvement of PP2A‐mediated tau dephosphorylation in alteration of EC MT structure and highlight the potential importance of PP2A in the regulation of EC the MT cytoskeleton and barrier function. © 2004 Wiley‐Liss, Inc.
The 33 kb transcript of the chicken α‐globin gene domain is part of the nuclear matrix
Authors: Razin Sergey V. , Rynditch Alla , Borunova Victoria , Ioudinkova Elena , Smalko Victor , Scherrer Klaus
Published: May 2004

Giant nuclear transcripts, and in particular the RNAs of the globin gene domains which are much larger than their canonical pre‐mRNAs, have been an enigma for many years. We show here that in avian erythroblastosis virus (AEV)‐transformed chicken erythroleukaemic cells, where globin gene expression is abortive, the whole domain of α‐globin genes is transcribed for about 33 kb in the globin direction and that this RNA is part of the nuclear matrix. Northern blot hybridisation with strand‐specific riboprobes, recognising genes and intergenic sequences, and RT‐PCR with downstream primers, show that the continuous full domain transcript (FDT) starts in the vicinity of a putative LCR and includes all the genes as well as known regulatory sites, the replication origin, and the DNA loop anchorage region in the upstream area. Absent in chicken fibroblasts, the globin FDT overlaps the major part of the ggPRX housekeeping gene that is transcribed in the opposite direction. RT‐PCR and in situ hybridisation with genic and extra‐genic globin probes demonstrated that the globin FDT is a component of the nuclear matrix. We suggest that the globin FDTs keep the domain in an active state, and the globin RNAs on the processing pathway are a component of the nuclear matrix. They may take part in the dynamic nuclear architecture when productively processed, or turn over slowly when globins are not synthesised. © 2004 Wiley‐Liss, Inc.
Regulation of HIF prolyl hydroxylases by hypoxia‐inducible factors
Authors: Aprelikova Olga , Chandramouli Gadisetti V.R. , Wood Matthew , Vasselli James R. , Riss Joseph , Maranchie Jodi K. , Linehan W. Marston , Barrett J. Carl
Published: May 2004

Hypoxia and induction of hypoxia‐inducible factors (HIF‐1α and HIF‐2α) is a hallmark of many tumors. Under normal oxygen tension HIF‐alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel‐Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re‐introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF‐1 and HIF‐2 and found that siRNA targeted degradation of HIF‐1α and HIF‐2α results in decreased hypoxia‐induced PHD3 expression. Ectopic overexpression of HIF‐2α in two different cell lines provided a much better induction of PHD3 gene than HIF‐1α. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF‐2α. However, induction of PHD2 by hypoxia has HIF‐1‐independent and ‐dependent components. Short‐term hypoxia (4 h) results in induction of PHD2 independent of HIF‐1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA‐mediated degradation of HIF‐1α subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation. Published 2004 Wiley‐Liss, Inc.
Mitogen‐activated protein kinases modulate H2O2‐induced apoptosis in primary rat alveolar epithelial cells
Authors: Carvalho Helotonio , Evelson Pablo , Sigaud Samuel , González‐Flecha Beatriz
Published: May 2004

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant‐mediated ATII cell apoptosis using in vitro models of H2O2‐stress. H2O2, delivered either as a bolus or as a flux, lead to time‐ and concentration‐dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H2O2 concentrations and production rates in the physiological range (1 μM) and peaked at 100 μM H2O2. Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H2O2 for maximal responses. H2O2‐induced apoptosis was preceded by rapid activation of all three classes of mitogen‐activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro‐apoptotic role for JNK pathway and an anti‐apoptotic role for ERK‐ and p38‐initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant‐related lung injury. © 2004 Wiley‐Liss, Inc.
K317, R319, and E320 within the proximal C‐terminus of the bradykinin B2 receptor form a motif important for phospholipase C and phospholipase A2 but not connective tissue growth factor related signaling
Authors: Yu Jun , Liu Bin , Eramian David , Mierke Dale , Taylor Linda , Polgar Peter
Published: May 2004

We showed previously that large domain exchanges between the bradykinin B2 (BKB2) and angiotensin II type 1a (AT1a) receptors can result in functional hybrids. However, when we proceeded to exchange the entire bradykinin B2 receptor (BKB2R) C‐terminal tail with the AT1aR C‐terminus, the hybrid, while continuing to bind BK and be endocytosed as wild type (WT) BKB2R, lost much of its ability to activate phosphatidylinositol (PI) turnover or the release of arachidonic acid (ARA). In this study, we constructed chimeric receptors within the proximal C‐terminus between the BKB2R and AT1aR or bradykinin B1 receptor (BKB1R). The mutant and WT receptor cDNAs were stably transfected into Rat‐1 cells. Also, point mutations were generated to evaluate the role of the individual residues within this region. These chimeric studies revealed that the proximal portion of the BKB2R C‐tail is crucial for G protein‐linked BKB2R functions. This region could not be swapped with the AT1aR to obtain a BK activated PI turnover or ARA release. Further studies demonstrated that the distal portion (325–330) of this region is exchangeable; however, the middle portion (317–324) is not. Small motif exchanges within this section identified the KSR and EVY motifs as crucial for Gαq, Gαi related signaling of the BKB2R. Point mutations then showed that the charged amino acids K317, R319, and E320 are the residues critical for linking to PI turnover and ARA release. However, these proximal chimeras showed normal receptor uptake. Interestingly, while apparently not activating G protein‐linked signaling, the proximal tail AT1aR exchange mutant and the entire C‐terminus exchange hybrid continued to cause a substantial bradykinin effected increase in connective tissue growth factor (CTGF) mRNA level, as WT BKB2R. © 2004 Wiley‐Liss, Inc.
Adrenal gland involvement in the regulation of renal 11β‐hydroxysteroid dehydrogenase 2
Authors: Zallocchi Marisa Laura , Matkovic Laura , Calvo Juan Carlos , Damasco María Cristina
Published: May 2004

Renal 11β‐hydroxysteroid dehydrogenase 2 (HSD2) catalyzes the conversion of active glucocorticoids to inert 11β‐keto compounds, thereby preventing the illicit binding of these hormones to mineralocorticoid receptors (MRs) and, thus, conferring aldosterone specificity. Absence or inhibition of HSD2 activity, originates a hypertensive syndrome with sodium retention and increased potassium elimination. Recent studies from our laboratory reported an increment of HSD2 activity in intact‐stressed rats. To evaluate the adrenal involvement in this increase, we analyzed HSD2 activity and protein abundance in Intact, Sham‐operated, and adrenalectomized rats under stress situations (gavage with an overload of 200 mM HCl (10 ml) and simulated gavage) or with corticosterone replacement. HSD2 activity was assessed in renal microsomal preparations obtained from different groups of animals. HSD2 protein abundance was measured by Western‐blot. Circulating corticosterone was determined by radioimmunoassay. Sham‐operated animals showed an increase in HSD2 activity and abundance compared to Intact and adrenalectomized rats suggesting the involvement of stress‐related adrenal factors in HSD2 regulation. In the case of acidotic adrenalectomized animals, there was an increase in renal HSD2 activity when, along with the HCl overload, the rats were injected with corticosterone. This increment occurred without an increase in enzyme abundance. These results suggest the importance of circulating levels of glucocorticoids to respond to a metabolic acidosis, through regulation of HSD2 stimulation. The group subjected to a simulated gavage showed an increase in enzyme activity and protein abundance, thus demonstrating the need for both adrenal and extra‐factors in the modulation of renal HSD2. The adrenalectomized animals injected with different doses of corticosterone, produced a progressive increase in enzyme activity and abundance, being significant for the dose of 68 μg corticosterone/100 g body weight. The highest dose (308 μg/100 g body weight) did not show any variation in activity and abundance compared to the control group. This biphasic effect of glucocorticoids could be explained taking into account their permissive and suppressive actions, depending on their blood levels. Knowing that stress induces multifactorial responses, it should not be suprising to observe a differential regulation in renal HSD2, confirming that different stressors act through different factors of both, adrenal and extra‐adrenal origin. © 2004 Wiley‐Liss, Inc.
Cellular senescence induced by p53‐ras cooperation is independent of p21waf1 in murine embryo fibroblasts
Authors: Castro Maria E. , Guijarro Maria del Valle , Moneo Victoria , Carnero Amancio
Published: May 2004

Oncogenic activation in primary murine fibroblasts initiates a senescence‐like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21‐null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53−/−;p21−/− double‐null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1‐independent mechanism. © 2004 Wiley‐Liss, Inc.
Functional domains of APOBEC3G required for antiviral activity
Authors: Li Jinliang , Potash Mary Jane , Volsky David J.
Published: May 2004

The viral protein, Vif, is essential for the production of infectious progeny virions in natural target cells of human immunodeficiency virus type 1 (HIV‐1). Several recent reports indicate that Vif acts by antagonizing the activity of an endogenous human antiviral protein, APOBEC3G. To investigate this route to restrict HIV‐1 infection, we employed mutagenesis to assess APOBEC3G function during HIV‐1 infection including interaction with Vif, localization, and activity in virions. We found that APOBEC3G binds Vif in infected cells and the C′‐terminal region is required for this interaction. APOBEC3G was only incorporated into virions in the absence of Vif and deletion of either the N′‐terminal or C′‐terminal regions of APOBEC3G abrogated virion localization. Assaying endogenous reverse transcription we found that APOBEC3G and its C′‐terminal deletion mutant inhibited full‐length cDNA synthesis, possibly through binding to viral RNA, a function revealed through gel‐shift assays. Taken together, our studies suggest that APOBEC3G inhibits HIV‐1 infection through interference with reverse transcription and that Vif counteracts APOBEC3G by impeding its entry into virions. © 2004 Wiley‐Liss, Inc.
N‐myristoyltransferase inhibitor protein is homologous to heat shock cognate protein 70
Authors: Selvakumar Ponniah , Lakshmikuttyamma Ashakumary , Pasha Mohammed Khysar , King Martin J. , Olson Douglas J.H. , Mori Sumiko , Ross Andrew R.S. , Hayashi Kiyoshi , Dimmock Jonathan R. , Sharma Rajendra K.
Published: May 2004

Many of viral and eukaryotic proteins are required for signal transduction and regulatory functions which undergo a lipid modification by the enzyme N‐myristoyltransferase (NMT). In this study, we demonstrated that heat shock cognate protein 70 (HSC70) is homologous to NMT inhibitor protein (NIP71), which was discovered in our laboratory, based on MALDI‐TOF mass spectrometric analysis. The purified bovine cytosolic HSC70 and particulate NIP71 produced a dose‐dependent inhibition of human NMT having half maximal inhibitions of 235 and 230 nM, respectively. Further, Western blot analysis revealed that the purified particulate NIP71 and cytosolic HSC70 cross‐reacted with both anti‐NIP71 and anti‐HSC70 antibodies. The results we obtained imply that molecular chaperones could be involved in the regulation of NMT in normal and cancerous cells. Further studies directed to revealing the role of HSC70 in the regulation of NMT may lead to the development of gene based therapies of colon cancer. © 2004 Wiley‐Liss, Inc.
Platelet‐derived growth factor‐induced arachidonic acid release for enhancement of prostaglandin E2 synthesis in human gingival fibroblasts pretreated with interleukin‐1β
Authors: Nakao Sumi , Ogata Yorimasa , Yamamoto Yoshifumi , Furuyama Shunsuke , Sugiya Hiroshi
Published: May 2004

Platelet‐derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E2 (PGE2) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time‐ and dose‐dependent manner, and simultaneously induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i), but less provoked PGE2 release and cyclooxygenase‐2 (COX‐2) mRNA expression. When [Ca2+]i was increased by Ca2+‐mobilizaing reagents, arachidonic acid release was increased. The PDGF‐induced arachidonic acid release and increase in [Ca2+]i were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre‐stimulated with interleukin‐1β (IL‐1β), PDGF clearly increased PGE2 release. The PDGF‐induced PGE2 release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL‐1β, arachidonic acid strongly enhanced PGE2 release and COX‐2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca2+]i via tyrosine kinase activation, and which contributes to PGE2 production via COX‐2 expression in HGF primed with IL‐1β. © 2004 Wiley‐Liss, Inc.
Differential effects of PD98059 and U0126 on osteogenesis and adipogenesis
Authors: Dang Zhi‐Chao , Lowik Clemens W.G.M.
Published: May 2004

PD98059 and U0126 are considered as specific inhibitors of the p42/44 mitogen‐activated protein kinases (MAPK) pathway, which affects osteogenesis and adipogenesis. Here, we show unexpected differential effects of PD98059 and U0126 on osteogenesis and adipogenesis as well as on estrogen (E2)‐induced actions in osteoprogenitor KS483 cells. PD98059 dose‐dependently inhibited osteogenesis indicated by cellular alkaline phosphatase (ALP) activity and nodule formation, but stimulated adipogenesis shown by the number of adipocytes. In contrast, U0126 slightly decreased osteogenesis but had no effects on adipogenesis, although it inhibited p42/44 MAPK more potently than PD98059. Furthermore, PD98059, but not U0126, counteracted E2‐induced osteogenesis and adipogenesis. Transfection experiments showed that PD98059, but not U0126, had estrogenic transcriptional activity. Interestingly, both PD98059 and U0126 potentiated E2‐induced estrogenic transcriptional activity in KS483 cells, which is opposite to the response in MCF7 breast cancer cells. Our data indicate that the cross‐talk between growth factors and estrogen receptor (ER)‐mediated pathways in KS483 cells is different from that in MCF7 cells. In summary, the differential effects of PD98059 and U0126 indicate their actions are not exclusively due to an inhibition of MAPK pathway. Caution should be taken in the interpretation of the results obtained using these inhibitors. © 2004 Wiley‐Liss, Inc.
Generation and metabolism of bioactive sphingosine‐1‐phosphate
Authors: Stunff Hervé Le , Milstien Sheldon , Spiegel Sarah
Published: May 2004

Sphingosine‐1‐phosphate (S1P) is a bioactive lysosphingophospholipid that has been implicated in the regulation of vital biological processes. Abundant evidence indicates that S1P acts as both an intracellular messenger and an extracellular ligand for a family of five specific G protein‐coupled S1P receptors (S1PRs). Cellular levels of S1P are tightly regulated in a spatio‐temporal manner through its synthesis catalyzed by sphingosine kinases (SphKs) and degradation by S1P lyase (SPL) and specific S1P phosphohydrolases. Over the past decade, the identification and cloning of genes encoding S1P metabolizing enzymes has increased rapidly. Overexpression and deletion of these enzymes has provided important insights into the intracellular and the “inside‐out” functions of S1P. The purpose of this review is to summarize the current knowledge of S1P metabolizing enzymes, their enzymatic properties, and their roles in the control of cellular functions by S1P. © 2004 Wiley‐Liss, Inc.
Regulation of biological activity of laminin‐5 by proteolytic processing of γ2 chain
Authors: Ogawa Takashi , Tsubota Yoshiaki , Maeda Masato , Kariya Yoshinobu , Miyazaki Kaoru
Published: May 2004

Laminin‐5 (LN5), which regulates both cell adhesion and cell migration, undergoes specific extracellular proteolytic processing at an amino‐terminal region of the γ2 chain as well as at a carboxyl‐terminal region of the α3 chain. To clarify the biological effect of the γ2 chain processing, we prepared a human recombinant LN5 with the 150‐kDa, non‐processed γ2 chain (GAA‐LN5) and natural LN5 with the 105‐kDa, processed γ2 chain (Nat‐LN5). Comparison of their biological activities demonstrated that GAA‐LN5 had an about five‐times higher cell adhesion activity but an about two‐times lower cell migration activity than Nat‐LN5. This implies that the proteolytic processing of LN5 γ2 chain converts the LN5 from the cell adhesion type to the cell migration type. It was also found that human gastric carcinoma cells expressing the LN5 with the non‐processed γ2 chain is more adherent but less migratory than the carcinoma cells expressing a mixture of LN5 forms with the processed γ2 chain and with the unprocessed one. The functional change of LN5 by the proteolytic processing of the γ2 chain may contribute to elevated cell migration under some pathological conditions such as wound healing and tumor invasion. © 2004 Wiley‐Liss, Inc.
Enhancement of immunogenicity of HPV16 E7 oncogene by fusion with E. coli β‐glucuronidase
Authors: Šmahel Michal , Pokorná Dana , Macková Jana , Vlasák Josef
Published: May 2004

Background

Human papillomavirus type 16 (HPV16) E7 is an unstable oncoprotein with low immunogenicity. In previous work, we prepared the E7GGG gene containing point mutations resulting in substitution of three amino acids in the pRb‐binding site of the HPV16 E7 protein.

Methods and results

To increase E7GGG immunogenicity we constructed fusion genes of E. coli β‐glucuronidase (GUS) with one or three copies of E7GGG. Furthermore, a similar construct was prepared with partial E7GGG (E7GGGp, 41 amino acids from the N‐terminus). The expression of the fusion genes was examined in human 293T cells. Quantification of GUS activity and the amount of E7 antigen showed substantially reduced GUS activity of fusion proteins with complete E7GGG that was mainly caused by decrease of their steady‐state level in comparison with GUS or E7GGGpGUS. Still, the steady‐state level of E7GGG.GUS was about 20‐fold higher than that of the E7GGG protein. The immunogenicity of the fusion genes with complete E7GGG was tested by DNA immunisation of C57BL/6 mice with a gene gun. TC‐1 cells and their clone TC‐1/A9 with down‐regulated MHC class I expression were subcutaneously (s.c.) inoculated to induce tumour formation. All mice were protected against challenge with TC‐1 cells and most animals remained tumour‐free in therapeutic‐immunisation experiments with these cells, in contrast to immunisation with unfused E7GGG and the fusion with the lysosome‐associated membrane protein 1 (Sig/E7GGG/LAMP‐1). Significant protection was also recorded against TC‐1/A9 cells. Both tetramer staining and ELISPOT assay showed substantially higher activation of E7‐specific CD8+ lymphocytes in comparison with E7GGG and Sig/E7GGG/LAMP‐1. Deletion of 231 bp in the GUS gene eliminated enzymatic activity, but did not influence the immunogenicity of the E7GGG.GUS gene.

Conclusions

The findings demonstrate the superior immunisation efficacy of the fusion genes of E7GGG with GUS when compared with E7GGG and Sig/E7GGG/LAMP‐1. The E7GGG.GUS‐based DNA vaccine might also be efficient against human tumour cells with reduced MHC class I expression. Copyright © 2004 John Wiley & Sons, Ltd.
Recombinant Sendai virus vectors deleted in both the matrix and the fusion genes: efficient gene transfer with preferable properties
Authors: Inoue Makoto , Tokusumi Yumiko , Ban Hiroshi , Shirakura Masayuki , Kanaya Takumi , Yoshizaki Mariko , Hironaka Takashi , Nagai Yoshiyuki , Iida Akihiro , Hasegawa Mamoru
Published: May 2004

Background

Sendai virus (SeV) is a new type of cytoplasmic RNA vector, which infects and replicates in most mammalian cells, directs high‐level expression of the genes on its genome and is free from genotoxicity. In order to improve this vector, both the matrix (M) and fusion (F) genes were deleted from its genome.

Methods

For the recovery of the M and F genes‐deleted SeV (SeV/ΔMΔF), the packaging cell line was established by using a Cre/loxP induction system. SeV/ΔMΔF was characterized and compared with wild‐type and F or M gene‐deleted SeV vectors in terms of transduction ability, particle formation, transmissible property and cytotoxicity.

Results

SeV/ΔMΔF was propagated in high titers from the packaging cell line. When this vector was administered into the lateral ventricle and the respiratory tissue, many of the ependymal and epithelial cells were transduced, respectively, as in the case of wild‐type SeV. F gene‐deletion made the SeV vector non‐transmissible, and M gene‐deletion worked well to inhibit formation of the particles from infected cells. Simultaneous deletions of these two genes in the same genome resulted in combining both advantages. That is, both virus maturation into particles and transmissible property were almost completely abolished in cells infected with SeV/ΔMΔF. Further, the cytopathic effect of SeV/ΔMΔF was significantly attenuated rather than that of wild type in vitro and in vivo.

Conclusions

SeV/ΔMΔF is an advanced type of cytoplasmic RNA vector, which retains efficient gene transfer, gains non‐transmissible properties and loses particle formation with less cytopathic effect. Copyright © 2004 John Wiley & Sons, Ltd.
Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer
Authors: Boeckle Sabine , von Gersdorff Katharina , van der Piepen Silke , Culmsee Carsten , Wagner Ernst , Ogris Manfred
Published: May 2004

Background

Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable.

Methods

Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice.

Results

SEC did not change the size and zeta‐potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8–15 µg/ml). At low DNA concentrations (2–4 µg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non‐purified particles or purified particles without free PEI.

Conclusions

This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose‐dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile. Copyright © 2004 John Wiley & Sons, Ltd.
Lentiviral vectors for treating and modeling human CNS disorders
Authors: Azzouz Mimoun , Kingsman Susan M. , Mazarakis Nicholas D.
Published: May 2004

Vectors based on lentiviruses efficiently deliver genes into many different types of primary neurons from a broad range of species including man and the resulting gene expression is long term. These vectors are opening up new approaches for the treatment of neurological diseases such as Parkinson's disease (PD), Huntington's disease (HD), and motor neuron diseases (MNDs). Numerous animal studies have now been undertaken with these vectors and correction of disease models has been obtained. Lentiviral vectors also provide a new strategy for in vivo modeling of human diseases; for example, the lentiviral‐mediated overexpression of mutated human α‐synuclein or huntingtin genes in basal ganglia induces neuronal pathology in animals resembling PD and HD in man. These vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non‐primate lentivirus, equine infectious anemia virus (EIAV). It will then describe some key examples of genetic correction and generation of genetic animal models of neurological diseases. The prospects for clinical application of lentiviral vectors for the treatment of PD and MNDs will also be outlined. Copyright © 2004 John Wiley & Sons, Ltd.
One‐pot synthesis of [14C]arsenobetaine bromide
Authors: Bernardo Martha , Francesconi Kevin , Kollenz Gert
Published: May 2004

A one‐pot synthesis of [14C]arsenobetaine from 2‐dimethylarsinoylacetic acid is described. Cation‐ and anion‐exchange columns are used in the purification of the compound and its conversion to the bromide salt. Copyright © 2004 John Wiley & Sons, Ltd.
Ultrasonic particle size reduction of radiolabeled pharmaceuticals
Authors: Schenk David J. , Jones Allen N. , Braun Matthew P. , Yao Dan , Wallace Michael A. , Marques Rosemary , Jenkins Herbert J. , Chang Anson , Jia Xiujuan , Crocker Louis S. , Dean Dennis C. , Melillo David G.
Published: May 2004

High‐intensity ultrasonic treatment of MeOH/H2O, IPA/H2O, EtOH or H2O suspensions of C‐14 labeled and unlabeled pharmaceuticals successfully reduced the average particle diameter from a range of 8–260 to 4–15 µm as determined by optical microscopy. Copyright © 2004 John Wiley & Sons, Ltd.
Synthesis of tritiated Cyclosporin A and FK‐506 by metal‐catalyzed hydrogen isotope exchange
Authors: Shevchenko V. P. , Nagaev I. Yu. , Myasoedov N. F. , Andres H. , Moenius T. , Susan A.
Published: May 2004

Tritium‐labeled Cyclosporin A (I) and FK‐506 (II) have been prepared using a metal‐catalysed hydrogen isotope exchange procedure and high specific activity tritiated water. Specific activities of the labeled compounds were 0.15 and 0.59 TBq/mmol, respectively. Copyright © 2004 John Wiley & Sons, Ltd.
First no‐carrier‐added radioselenation of an adenosine‐A1 receptor ligand
Authors: Blum Till , Ermert Johannes , Wutz Walter , Bier Dirk , Coenen Heinz H.
Published: May 2004

The precursor synthesis and the no‐carrier‐added (n.c.a.) radiosynthesis of the adenosine‐A1 receptor ligand 5′‐(methyl[75Se]seleno)‐N6‐cyclopentyladenosine ([75Se]1) are described in this report. A method was developed starting from elemental n.c.a. selenium‐75, followed by a three‐step polymer‐supported radioselenation and deprotection which gave the radioligand with a radiochemical yield of 30%, a radiochemical purity of > 99% and a specific radioactivity of > 300 GBq/mmol (8 Ci/mmol). Preparation time was 40 min. The nonradioactive compound 5′‐(methylseleno)‐N6‐cyclopentyladenosine (1) was pharmacologically evaluated in vitro and showed high affinity and selectivity for the adenosine‐A1 receptor. These preliminary results suggest that this compound could be a useful radioligand for the noninvasive imaging of the brain adenosine‐A1 receptors using positron emission tomography (PET) when labelled with the positron emitter selenium‐73 (half‐life: 7.1 h). Copyright © 2004 John Wiley & Sons, Ltd.
Comparison of pathways to the versatile synthon of no‐carrier‐added 1‐bromo‐4‐[18F]fluorobenzene
Authors: Ermert Johannes , Hocke Carsten , Ludwig Thomas , Gail René , Coenen Heinz H.
Published: May 2004

The availability of no‐carrier‐added (n.c.a.) 1‐bromo‐4‐[18F]fluorobenzene with high radiochemical yields is important for 18F‐arylation reactions using metallo‐organic 4‐[18F]fluorophenyl compounds (e.g. of lithium or magnesium) or Pd‐catalyzed coupling. In this study, different methods for the preparation of 1‐bromo‐4‐[18F]fluorobenzene by nucleophilic aromatic substitution reactions using n.c.a. [18F]fluoride were examined. Of six pathways compared, symmetrical bis‐(4‐bromphenyl)iodonium bromide proved most useful to achieve the title compound in a direct, one‐step nucleophilic substitution with a radiochemical yield (RCY) of 65% within 10 min. Copyright © 2004 John Wiley & Sons, Ltd.
Simultaneous determination of mono‐, di‐ and tributyltin in environmental samples using isotope dilution gas chromatography mass spectrometry
Authors: Centineo Giuseppe , Rodríguez‐González Pablo , González Elisa Blanco , García Alonso J. Ignacio , Sanz‐Medel Alfredo
Published: May 2004

The development of a rapid, precise and accurate speciation method for the simultaneous determination of mono‐, di‐ and tributyltin in environmental samples is described. The method is based on using isotope dilution gas chromatography/mass spectrometry (GC/MS) with electron ionization, a widely used technique in routine testing laboratories. A mixed spike containing 119Sn‐enriched monobutyltin (MBT), dibutyltin (DBT) and tributyltin (TBT) was used for the isotope dilution of the samples. Five molecular ions were monitored for each analyte, corresponding to the 116Sn, 117Sn, 118Sn, 119Sn and 120Sn isotopes. The detection at masses corresponding to 116Sn and 117Sn were used to correct for m + 1 and m + 2 contributions of 13C from the organic groups attached to the tin atom on the 118Sn, 119Sn and 120Sn masses with simple mathematical equations and the concentrations of the butyltin compounds were calculated based on the corrected 118Sn/119Sn and 120Sn/119Sn isotope ratios. The 119Sn‐enriched multispecies spike was applied with satisfactory results to the simultaneous determination of MBT, DBT and TBT in three certified reference materials: two sediments, PACS‐2 and BCR 646, and the mussel tissue CRM 477. The method was compared with a previously published GC/inductively coupled plasma MS isotope dilution procedure, developed in our laboratory, by injecting the same samples into both instruments. Comparable analytical results in terms of precision and accuracy are demonstrated for both atomic and molecular mass spectrometric detectors. Thus, reliable quantitative organotin speciation analysis can be achieved using the more widespread and inexpensive GC/MS instrument. Copyright © 2004 John Wiley & Sons, Ltd.
Simultaneous quantitative analysis of isobars by tandem mass spectrometry from unresolved chromatographic peaks
Authors: Kushnir Mark M. , Rockwood Alan L. , Nelson Gordon J.
Published: May 2004

A method was developed for the simultaneous quantitation of isobars from unresolved chromatographic peaks. The method is based on differences in branching ratios of ion abundances in their tandem mass spectra and an assumption that the product ion mass spectra of a mixture can be considered as a linear combination of the spectra of individual constituents. We present analytical equations and a matrix‐based approach for deconvoluting the concentration of individual components from the total peak intensity for two and three isobars and also a matrix‐based generalization to any number of compounds. The feasibility of the simultaneous analysis of mixtures containing two compounds was assessed. The approach was evaluated for the analysis of structural isomers of methylmalonic and succinic acids in human plasma and urine samples for a group of 270 samples. The linear regression equation, standard error and correlation coefficient for the agreement with a traditional method utilizing chromatographic separation of the isomers were y = 0.999x − 0.005, 0.024 µmol l−1, and 0.985, respectively. The utility of a spectral contrast angle as a predictor of analysis feasibility was evaluated. Copyright © 2004 John Wiley & Sons, Ltd.
Mass spectrometry of bisquinolizidine alkaloids: 2‐ and 17‐cyano‐substituted derivatives of sparteine and lupanine
Authors: Jasiewicz Beata , Boczoń Władysław , Wyrzykiewicz Elżbieta
Published: May 2004

The mass spectral fragmentations of 2‐cyano‐2‐methylsparteine (1), 2‐cyano‐2‐phenylsparteine (2), 2‐cyano‐2‐(p‐tolyl)sparteine (3), 17‐cyanosparteine (4) and 17‐cyanolupanine (5) were investigated. Fragmentation pathways, whose identification was assisted by accurate mass measurements and a correlation between the abundances of the M+• and the selected fragment ions of the investigated compounds, are discussed. The data obtained create the basis for distinguishing the structural metamers. Copyright © 2004 John Wiley & Sons, Ltd.
Dissociation of ionized benzophenones investigated by the kinetic method: effective temperature, steric effects and gas‐phase CO+• affinities of phenyl radicals
Authors: Augusti Rodinei , Turowski Maciej , Chen Hao , Graham Cooks R.
Published: May 2004

Ionized benzophenones ([PhC(O)PhY]+•; Y = 4 − NO2, 4 − CF3, 4‐F, 4‐Br, 4‐Me, 3,4‐diMe, 4‐OH, 4‐OMe, 2‐Cl, 2‐Me, 2‐OH, 2,6‐diMe) undergo competitive dissociation upon collision‐induced dissociation (CID) at 20 eV collision energy to generate benzoyl cations ([PhCO]+ and [YPhCO]+) and phenyl radicals (Ph• and YPh•). For the para‐substituted benzophenones, the natural logarithm of the abundance ratio of the benzoyl cations [ln([PhCO+]/[YPhCO+])] is found to correlate linearly with the calculated CO+• affinities of the phenyl radicals Ph• and YPh•. A deviation from linearity is observed for the ortho‐substituted isomers. This is probably due to a significant intramolecular steric interaction between the carbonyl group and the ortho substituent which prevents the formation of a stable planar system. An observed shift in the intercept relative to the origin is interpreted as the result of a systematic error in the calculated CO+• affinities and this effect is minimized by calculations at a higher level. The dissociation of ionized para‐substituted benzophenones is associated with a relatively high effective temperature of 1816 ± 41 K, calculated from the slope of the kinetic method plot, a value that is consistent with a covalent bond in the activated ion. In addition, Δ(ΔS), the dissociation entropy of the benzoyl cations to form CO+• and the aryl radical, is found to be about 4 J mol−1 K−1 by employing the extended version of the kinetic method. Copyright © 2004 John Wiley & Sons, Ltd.
A digital ion trap mass spectrometer coupled with atmospheric pressure ion sources
Authors: Ding Li , Sudakov Michael , Brancia Francesco L. , Giles Roger , Kumashiro Sumio
Published: May 2004

In a digital ion trap (DIT), the quadrupole trapping and excitation waveforms are generated by the rapid switching between discrete d.c. voltage levels. As the timing of the switch can be controlled precisely by digital circuitry, the approach provides an opportunity to generate mass spectra by means of a frequency scan in contrast to the conventional voltage scan, thus providing a wider mass range of analysis. An instrument has been constructed which employs a ‘non‐stretched’ ion trap and the field fault around the aperture of the end‐cap electrode can be corrected electronically using a field‐adjusting electrode. The ion trap was coupled with electrospray ionization (ESI) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) sources to demonstrate the capability of the digital method. AP‐MALDI mass spectra of singly charged ions with mass‐to‐charge ratios upto 17 000 Th were generated using a trapping voltage of only 1000 V. Forward and reverse mass scans at resolutions up to 19 000 and precursor ion isolation at resolutions up to 3500 with subsequent tandem mass spectrometric analysis were demonstrated. The method of generating the digital waveforms and period scan is described. Discussion of the issues of mass range, scan speed, ion trapping efficiency and collision‐induced dissociation efficiency are also provided. Copyright © 2004 John Wiley & Sons, Ltd.
Neuronal NOS activation during oxygen and glucose deprivation triggers cerebellar granule cell death in the later reoxygenation phase
Authors: Scorziello A. , Pellegrini C. , Secondo A. , Sirabella R. , Formisano L. , Sibaud L. , Amoroso S. , Canzoniero L.M.T. , Annunziato L. , Di Renzo G.F.
Published: May 2004

The present study investigated the temporal relationship between neuronal nitric oxide synthase (nNOS) activity and expression and the development of neuronal damage occurring during anoxia and anoxia followed by reoxygenation. For this purpose, cerebellar granule cells were exposed to 2 hr of oxygen and glucose deprivation (OGD) and 24 hr of reoxygenation. To clarify the consequences of nNOS activity inhibition on neuronal survival, cerebellar granule cells were exposed to OGD, both in the absence of extracellular Na+ ([Na+]e), a condition that by reducing intracellular Ca2+ ([Ca2+]I) prevents Ca2+‐dependent nNOS activation, and in the presence of selective and nonselective nNOS inhibitors, such as Nω‐L‐allyl‐L‐arginine (L‐ALA), Nω‐propyl‐L‐arginine (NPLA), and L‐nitro‐arginine‐methyl‐ester (L‐NAME), respectively. The results demonstrated that the removal of [Na+]e hampered the [Ca2+]i increase and decreased expression and activity of nNOS. Similarly, the increase of free radical production present in cerebellar neurons, exposed previously to OGD and OGD/reoxygenation, was abolished completely in the absence of [Na+]e. Furthermore, the absence of [Na+]e in cerebellar neurons exposed to 2 hr of OGD led to the improvement of mitochondrial activity and neuronal survival, both after the OGD phase and after 24 hr of reoxygenation. Finally, the exposure of cerebellar neurons to L‐ALA (200 nM), and L‐NAME (500 μM) was able to effectively reduce NO• production and caused an increase in mitochondrial oxidative activity and an improvement of neuronal survival not only during OGD, but also during reoxygenation. Similar results during OGD were obtained also with NPLA (5 nM), another selective nNOS inhibitor. These data suggest that the activation of nNOS is highly accountable for the neuronal damage occurring during the OGD and reoxygenation phases. © 2004 Wiley‐Liss, Inc.
Characteristics of the trigeminal depressor response in cats
Authors: Ohshita Naohiro , Nakajo Nobuyoshi , Takemura Motohide
Published: May 2004

We studied the effects of electrical stimulation of the inferior alveolar nerve (IAN) on cardiovascular responses in cats. There was statistical correlation between cardiovascular response and prestimulus mean arterial blood pressure (MABP) and heart rate (HR). A trigeminal depressor response (TDR) was induced when the prestimulus MABP and HR were above 95 mm Hg and 140 beats/min, respectively. We investigated further to identify the vasomotor regulating center and neural transmitters involved in TDR. In the medulla, electrical stimulation of the dorsomedial medulla, the infratrigeminal nucleus (IFT), and the rostral ventrolateral medulla (RVLM) induced a vasopressor response. We confirmed that neurons in the RVLM were retrogradely labeled by wheat germ agglutinin‐conjugated horseradish peroxidase injection into the nucleus intermediolateralis of the spinal cord. The vasopressor response induced by IFT stimulation was similar to that induced by IAN stimulation. Vasodepressor responses were induced when the caudal ventrolateral medulla, the nucleus tractus solitarius, the lateral tegmental field, the trigeminal nucleus interpolaris, the trigeminal spinal tract, and the paramedian reticular nucleus were stimulated. These responses, however, were not similar to the vasodepressor response induced by IAN stimulation but were similar to the cardiovascular response induced by vagal afferent stimulation. After spinalization or lesion of the RVLM, MABP and HR decreased and TDR completely disappeared. Inhibitory synaptic ligands and receptors were localized using immunohistochemical techniques. Neurons immunopositive for adrenaline, noradrenaline, and γ‐aminobutyric acid (GABA), and adrenaline α2A, GABAA, GABAB, and glycine receptors were distributed along the sympatho‐reflexive route including the RVLM and IFT. These results suggest that TDR could be induced as negative feedback to sympathetic hyperactivity whenever MABP and HR are high, because of the inhibitory control of the RVLM. © 2004 Wiley‐Liss, Inc.
Calcium signal‐mediated expression of the vasoactive intestinal polypeptide gene and its small contribution to activity‐dependent survival of mouse cerebellar granule cells
Authors: Fukuchi Mamoru , Sakuragawa Saori , Tabuchi Akiko , Tsuda Masaaki
Published: May 2004

We have demonstrated previously in primary cultures of mouse cerebellar granule cells (CGCs) that endogenously synthesized pituitary adenylate cyclase‐activating polypeptide (PACAP) contributes at least in part to the activity‐dependent survival of CGCs (Tabuchi et al. [2001] Neurosci. Res. 39:85–93). In this study, we have demonstrated that expression of vasoactive intestinal polypeptide (VIP), a member of the same VIP/secretin/glucagon family as PACAP, was activated markedly by Ca2+ influx through L‐type voltage‐dependent Ca2+ channels (L‐VDCCs), which could be induced under the depolarizing condition induced by high concentration of potassium (K+) in the medium. The activation of VIP mRNA expression, different from that of PACAP, was dependent partly on de novo protein synthesis. On the other hand, mRNA expression of secretin and PACAP/VIP receptors (PAC1, VPAC1, and VPAC2) was not activated by the Ca2+ influx; rather, PAC1 mRNA expression was reduced. Exogenously added VIP prevented apoptosis of CGCs under nondepolarizing conditions, the effect of which was mediated specifically through the VPAC1 receptor. Furthermore, the survival of CGCs under depolarizing conditions could be mediated partly through VPAC1, the contribution of which was much less than that of PAC1. These findings indicate that PACAP and VIP genes are coordinately activated by the Ca2+ signals in CGCs, but the contribution of VIP to the activity‐dependent survival of CGCs is quite small. © 2004 Wiley‐Liss, Inc.
Localization of wild‐type and mutant neuronal ceroid lipofuscinosis CLN8 proteins in non‐neuronal and neuronal cells
Authors: Lonka Liina , Salonen Tarja , Siintola Eija , Kopra Outi , Lehesjoki Anna‐Elina , Jalanko Anu
Published: May 2004

Neuronal ceroid lipofuscinoses (NCLs) are a group of childhood‐onset neurodegenerative disorders characterized by accumulation of autofluorescent lipopigment in many tissues, especially in neurons. Mutations in the CLN8 gene underlie Northern epilepsy (progressive epilepsy with mental retardation [EPMR], OMIM 600143) and a subset of Turkish variant late infantile NCL, but the pathogenetic mechanisms have remained elusive. The CLN8 transmembrane protein is an endoplasmic reticulum (ER) resident protein that recycles between ER and ER‐Golgi intermediate compartment (ERGIC) in non‐neuronal cells. To explore the disease mechanisms, we have characterized the neuronal localization of wild‐type CLN8 protein as well as CLN8 proteins representing patient mutations. Semliki Forest virus‐mediated CLN8 protein localized in the ER of mouse hippocampal primary neurons when compared to subcellular markers by immunofluorescence analysis. We also analyzed the possible polarized targeting of CLN8 and observed basolateral targeting in polarized epithelial CaCo‐2 cells, suggesting that CLN8 may locate outside the ER or in a specialized subcompartment of the ER. We were not able, however, to demonstrate differential distribution of CLN8 between axons and dendrites of neurons. Fractionation of mouse brain tissue indicated that endogenous mouse Cln8 is observed in light membrane fractions, different from ER, which further suggested differential localization for CLN8 in polarized cells. The disease mutations did not affect intracellular localization of CLN8 in non‐neuronal or neuronal cells. Consequently, there is no obvious genotype–phenotype correlation at the level of protein localization and thus mutations most likely directly affect functionally important domains of CLN8. © 2004 Wiley‐Liss, Inc.