bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomic fingerprints distinguish microglia, bone marrow, and spleen macrophage populations
Authors: Enose Yoshimi , Destache Christopher J. , Mack Andrea L. , Anderson James R. , Ullrich Fred , Ciborowski Pawel S. , Gendelman Howard E.
Published: March 2005

Mononuclear phagocytes (MP; dendritic cells, monocytes, tissue macrophages, and microglia) maintain tissue homeostasis and provide a first line of defense against invading pathogens. In specific circumstances, MPs also induce inflammatory responses and as such affect disease onset and progression. Despite intensive research into MP biology, little is known of the functional and molecular properties of individual MP subtypes. Using a novel proteomics platform, unique protein patterns and protein identities were observed among populations of spleen and bone marrow macrophages and microglia. Cells were obtained from C57BL/6 mice and were cultivated in macrophage colony‐stimulating factor. MP subtypes were indistinguishable by morphological or antigenic criteria. Protein profiling by Surface Enhanced Laser Desorption Ionization‐Time of Flight (SELDI‐TOF) ProteinChip® assays with weak cationic exchange chips showed unique MP spectral profiles. Corresponding protein fractions were recovered by high performance liquid chromatography and identified by liquid chromatography tandem mass spectrometry. The results provide a unique means to distinguish microglia from other MP subtypes. © 2005 Wiley‐Liss, Inc.
Inhibition of cysteine protease activity disturbs DNA replication and prevents mitosis in the early mitotic cell cycles of sea urchin embryos
Authors: Concha Carolina , Monardes Antonia , Even Yasmine , Morin Violeta , Puchi Marcia , Imschenetzky Maria , Marie Genevière Anne
Published: March 2005

Recent findings suggested that the role of cysteine proteases would not be limited to protein degradation in lysosomes but would also play regulatory functions in more specific cell mechanisms. We analyzed here the role of these enzymes in the control of cell cycle during embryogenesis. The addition of the potent cysteine protease inhibitor E64d to newly fertilized sea urchin eggs disrupted cell cycle progression, affecting nuclear as well as cytoplasmic characteristic events. Monitoring BrdU incorporation in E64d treated eggs demonstrated that DNA replication is severely disturbed. Moreover, this drug treatment inhibited male histones degradation, a step that is necessary for sperm chromatin remodeling and precedes the initiation of DNA replication in control eggs. This inhibition likely explains the DNA replication disturbance and suggests that S phase initiation requires cysteine protease activity. In turn, activation of the DNA replication checkpoint could be responsible for the consecutive block of nuclear envelope breakdown (NEB). However, in sea urchin early embryos this checkpoint doesn't control the mitotic cytoplasmic events that are not tightly coupled with NEB. Thus the fact that microtubule spindle is not assembled and cyclin B‐cdk1 not activated under E64d treatment more likely rely on a distinct mechanism. Immunofluorescence experiments indicated that centrosome organization was deficient in absence of cysteine protease activity. This potentially accounts for mitotic spindle disruption and for cyclin B mis‐localization in E64d treated eggs. We conclude that cysteine proteases are essential to trigger S phase and to promote M phase entry in newly fertilized sea urchin eggs. © 2005 Wiley‐Liss, Inc.
Molecular mechanisms of glutamine action
Authors: Curi R. , Lagranha C.J. , Doi S.Q. , Sellitti D.F. , Procopio J. , Pithon‐Curi T.C. , Corless M. , Newsholme P.
Published: March 2005

Glutamine is the most abundant free amino acid in the body and is known to play a regulatory role in several cell specific processes including metabolism (e.g., oxidative fuel, gluconeogenic precursor, and lipogenic precursor), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, contractile protein mass, redox potential, respiratory burst, insulin resistance, insulin secretion, and extracellular matrix (ECM) synthesis. Glutamine has been shown to regulate the expression of many genes related to metabolism, signal transduction, cell defense and repair, and to activate intracellular signaling pathways. Thus, the function of glutamine goes beyond that of a simple metabolic fuel or protein precursor as previously assumed. In this review, we have attempted to identify some of the common mechanisms underlying the regulation of glutamine dependent cellular functions. © 2005 Wiley‐Liss, Inc.
Enhanced angiogenesis characteristic of SPARC‐null mice disappears with age
Authors: Reed May J. , Bradshaw A.D. , Shaw M. , Sadoun E. , Han N. , Ferara N. , Funk S. , Puolakkainen P. , Sage E.H.
Published: March 2005

The impairment of angiogenesis in aging has been attributed, in part, to alterations in proteins associated with the extracellular matrix (ECM). SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM‐40) is a matricellular protein that regulates endothelial cell function as well as cell–ECM interactions. We have previously shown that angiogenesis, as reflected by fibrovascular invasion into subcutaneously implanted polyvinyl alcohol (PVA) sponges, is increased in SPARC‐null mice (6–9 months of age) relative to their wild‐type (WT) counterparts. In this study, we define the influence of aging on (a) the expression of SPARC and (b) fibrovascular invasion into sponge implants in SPARC‐null and WT mice. The expression of SPARC in fibroblasts and endothelial cells derived from young donors (humans mean age less than 30 years and mice 4–6 months of age) and old donors (humans mean age over 65 years and mice 22–27 months of age) decreased 1.6 to 2.3‐fold with age. Analysis of fibrovascular invasion into sponges implanted into old (22–27 months) SPARC‐null and WT mice showed no differences in percent area of invasion or collagenous ECM. Moreover, sponges from old SPARC‐null and WT mice contained similar levels of VEGF that were significantly lower than those from young (4–6 months) mice. In contrast to fibroblasts from young SPARC‐null mice, dermal fibroblasts from old SPARC‐null mice did not migrate farther, proliferate faster, or produce greater amounts of VEGF relative to their old WT counterparts. However, when stimulated with TGF‐β1, primary cells isolated from the sponge implants, and dermal fibroblasts from both old SPARC‐null and WT mice, showed marked increases in VEGF secretion. These data indicate that aging results in a loss of enhanced angiogenesis in SPARC‐null mice, as a result of the detrimental impact of age on cellular functions, collagen deposition, and VEGF synthesis. However, the influence of aging on these processes may be reversed, in part, by growth factor stimulation. © 2005 Wiley‐Liss, Inc.
Inhibition of Cdk6 expression through p38 MAP kinase is involved in differentiation of mouse prechondrocyte ATDC5
Authors: Moro Toru , Ogasawara Toru , Chikuda Hirotaka , Ikeda Toshiyuki , Ogata Naoshi , Maruyama Zenjiro , Komori Toshihisa , Hoshi Kazuto , Chung Ung‐Il , Nakamura Kozo , Okayama Hiroto , Kawaguchi Hiroshi
Published: March 2005

Because a temporal arrest in the G1‐phase of the cell cycle is a prerequisite for cell differentiation, this study investigated the involvement of cell cycle factors in the differentiation of cultured mouse prechondrocyte cell line ATDC5. Among the G1 cell cycle factors examined, both protein and mRNA levels of cyclin‐dependent kinase (Cdk6) were downregulated during the culture in a differentiation medium. The protein degradation of Cdk6 was not involved in this downregulation because proteasome inhibitors did not reverse the protein level. When inhibitors of p38 MAPK, ERK‐1/2, and PI3K/Akt were added to the culture, only a p38 MAPK inhibitor SB203580 blocked the decrease in the Cdk6 protein level by the differentiation medium, indicating that the Cdk6 inhibition was mediated by p38 MAPK pathway. In fact, p38 MAPK was confirmed to be phosphorylated during differentiation of ATDC5 cells. Enforced expression of Cdk6 in ATDC5 cells blocked the chondrocyte differentiation and inhibited Sox5 and Sox6 expressions. However, the Cdk6 overexpression did not affect the proliferation or the cell cycle progression, suggesting that the inhibitory effect of Cdk6 on the differentiation was exerted by a mechanism largely independent of its cell cycle regulation. These results indicate that Cdk6 may be a regulator of chondrocyte differentiation and that its p38‐mediated downregulation is involved in the efficient differentiation. © 2005 Wiley‐Liss, Inc.
Docosahexaenoic acid induces apoptosis in proliferating human endothelial cells
Authors: Kim Hyo Jung , Vosseler Claudia A. , Weber Peter C. , Erl Wolfgang
Published: March 2005

n‐3 polyunsaturated fatty acids (PUFAs) have been shown to exert beneficial effects in the prevention of cardiovascular disease, inflammation, and on tumor growth. To investigate effects of PUFAs on proliferation and apoptosis in endothelial cells, we tested the n‐3 PUFA docosahexaenoic acid (DHA) and the n‐6 PUFA arachidonic acid (AA) in human umbilical vein endothelial cells (HUVEC). The mitochondrial membrane potential (MMP) and the production of reactive oxygen species were examined by flow cytometry. Phosphorylation of p53 or p38 MAP kinase, and total levels of p53 were measured by Western blot. DNA binding activity of p53 was analyzed with a TransAM transcription factor assay kit. Tube formation was assessed on Matrigel. In proliferating HUVEC, but not in confluent cells, DHA reduced cell viability and induced apoptosis, as demonstrated by increases in membrane leakage (propidium iodide (PI) staining), Annexin‐V binding, sub G1 phase in the cell cycle, and TUNEL‐positive cells. AA had no effect on these parameters. In addition to a reduced MMP and increased reactive oxygen species, phosphorylation of p38 and p53 (serine 15) and impaired DNA binding of p53 were observed. There was no change in total levels of p53. The p38 inhibitor SB203580 had no effect on Annexin V binding. DHA also attenuated HUVEC tube formation. Taken together, DHA induces apoptosis in proliferating, but not in resting HUVEC, potentially via the phosphorylation of p53, resulting in decreased p53 DNA binding. The results suggest that anti‐angiogenic effects of DHA may be due to induction of apoptosis in proliferating endothelial cells. © 2005 Wiley‐Liss, Inc.
Dominant expression of androgen receptors and their functional regulation of organic anion transporter 3 in rat brain capillary endothelial cells; Comparison of gene expression between the blood–brain and ‐retinal barriers
Authors: Ohtsuki Sumio , Tomi Masatoshi , Hata Toshio , Nagai Yoko , Hori Satoko , Mori Shinobu , Hosoya Ken‐ichi , Terasaki Tetsuya
Published: March 2005

Brain and retinal capillary endothelial cells (BCECs and RCECs, respectively) exhibit a barrier structure and function. Comparison of gene expression in these cells could clarify the selective function of each barrier. The purpose of this study was to identify the genes selectively expressed at the blood–brain barrier (BBB) and to clarify the function of the selective gene, androgen receptor (AR). Gene expression was compared by a differential display using conditionally immortalized rat BCECs and RCECs (TR‐BBB and TR‐iBRB, respectively). A total of 12 gene fragments were identified as the selective genes dominantly expressed in TR‐BBB cells. The most selective fragment in TR‐BBB cells had the highest homology with the 3′‐UTR of human and mouse AR. Rat AR mRNA was detected in TR‐BBB cells and the brain capillary rich fraction, but not in TR‐iBRB cells. Expression of organic anion transporter 3 (OAT3) mRNA in TR‐BBB cells was induced by treatment with dihydrotestosterone (DHT), an AR ligand, and this induction was suppressed by flutamide. Moreover, uptake of benzylpenicillin by TR‐BBB cells was also induced by DHT treatment. In contrast, OAT3 mRNA expression in TR‐iBRB cells was not affected by DHT treatment. The brain‐to‐blood efflux rate of benzylpenicillin was not affected by gender. These results suggest that AR is involved in the functional regulation of OAT3 at the BBB, but not at the inner blood‐retinal barrier (iBRB), and this regulation is not affected by gender. The BBB function will be affected by the androgen levels in the brain and/or plasma via AR. © 2005 Wiley‐Liss, Inc.
BT‐IgSF, a novel immunoglobulin superfamily protein, functions as a cell adhesion molecule
Authors: Harada Hideki , Suzu Shinya , Hayashi Yokichi , Okada Seiji
Published: March 2005

BT‐IgSF is a newly identified cell surface glycoprotein belonging to the immunoglobulin superfamily (IgSF). We have previously shown that the expression of the BT‐IgSF gene was highly restricted to brain and testis, and its transcript was detected in both neurons and glial cells. In this study, to explore its function, we generated cells overexpressing BT‐IgSF proteins and analyzed their phenotypes. We found that the constitutive expression of BT‐IgSF in the myeloid leukemia cell line TF‐1‐fms did not alter the growth rates, but caused the formation of large cell aggregates. The cell aggregates were also observed with mutant BT‐IgSF lacking its cytoplasmic tail, the amino acid sequences of which were highly conserved among the BT‐IgSF subgroup proteins. The neutralizing antibody to β1 integrin did not diminish the cell aggregate formation. These results indicate that BT‐IgSF functions as a cell adhesion molecule, that its cytoplasmic tail is not essential for the function, and that β1 integrin is not involved in the function. We confirmed the cell adhesion function using NIH/3T3 fibroblastic cells expressing BT‐IgSF in an inducible system. Flow cytometric analyses with the cells demonstrated that the cell aggregation mediated by BT‐IgSF was through homophilic molecular interaction, and in a Ca2+/Mg2+‐independent manner. Coupled with its restricted pattern of the expression, the cell adhesion‐inducing function of BT‐IgSF suggests a role of the cell surface molecule in the development/function of the central nervous system and spermatogenesis. © 2005 Wiley‐Liss, Inc.
Coloring graphs from lists with bounded size of their union
Authors: Král' Daniel , Sgall Jiří
Published: March 2005

A graph G is k‐choosable if its vertices can be colored from any lists L(ν) of colors with |L(ν)| ≥ k for all ν ∈ V(G). A graph G is said to be (k,ℓ)‐choosable if its vertices can be colored from any lists L(ν) with |L(ν)| ≥k, for all ν∈ V(G), and with $|\bigcup_{{\nu}\in V(G)}\, L(\nu)|\le \ell$. For each 3 ≤ k ≤ ℓ, we construct a graph G that is (k,ℓ)‐choosable but not (k,ℓ + 1)‐choosable. On the other hand, it is proven that each (k,2k − 1)‐choosable graph G is O(k · ln k · 24k)‐choosable. © 2005 Wiley Periodicals, Inc. J Graph Theory
Validated toxicological determination of 30 drugs of abuse as optimized derivatives in oral fluid by long column fast gas chromatography/electron impact mass spectrometry
Authors: Gunnar Teemu , Ariniemi Kari , Lillsunde Pirjo
Published: April 2005

An analytical procedure was developed for the simultaneous sensitive identification, screening and quantitation of 30 drugs of abuse using 250 µl of human oral fluid. The method employs sequential mixed‐mode solid‐phase extraction (SPE), optimized derivative formation and long‐column fast gas chromatography/electron impact mass spectrometry (GC/EI‐MS). After sequential SPE elution, the most sensitive and stable derivatives were formed by taking careful account of the characteristics of the active functional groups and possible steric hindrances affecting derivatization chemistry. Amphetamine‐type stimulant drugs were acylated with heptafluorobutyric anhydride, benzodiazepines and Δ9‐tetrahydrocannabinol were silylated with N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide and benzoylecgonine, codeine, ethylmorphine, 6‐monoacetylmorphine, morphine, pholcodine, buprenorphine and norbuprenorphine with N‐methyl‐N‐(trimethylsilyl)trifluoroacetamide. In addition, the following analytes were included: methadone, cocaine, alprazolam, midazolam, fentanyl and zolpidem. In GC separation, fast temperature ramping and high carrier gas flow‐rate combined with long 30 m columns of i.d. 0.32 mm offered a reduction in analysis time and sharp peak shapes while still maintaining sufficient resolution and high sample capacity. Validated parameters including selectivity, linearity, accuracy, intra‐ and inter‐day precision, extraction efficiency and limit of quantitation were all within required limits. In contrast to previously published methods, this single procedure is suitable for the simultaneous toxicological determination of the most common illicit drugs and benzodiazepines, and also zolpidem, in a small amount of oral fluid. Copyright © 2005 John Wiley & Sons, Ltd.
Temporal profiling of gene expression during neurogenesis and remodeling in the olfactory epithelium at short intervals after target ablation
Authors: Getchell Thomas V. , Liu Hua , Vaishnav Radhika A. , Kwong Kevin , Stromberg Arnold J. , Getchell Marilyn L.
Published: March 2005

Neurogenesis in the olfactory epithelium (OE) is induced by olfactory bulbectomy (OBX), which effectively axotomizes olfactory sensory neurons (OSNs) and removes their synaptic targets, resulting in apoptosis. We used Affymetrix high‐density oligonucleotide arrays to investigate changes in gene expression during initiation of signaling in pathways that regulate apoptosis and neurogenesis in the murine OE at 2, 8, 16, and 48 hr after bilateral OBX compared to that in sham‐operated controls. We focused on regulation of a defined set of genes associated with apoptosis, stem/progenitor cell regulation, and cell cycle progression because of the activation of these processes in OE degeneration and remodeling after OBX. After data scrubbing and categorical analysis, one‐way analysis of variance identified 72 genes (4.9% of the present known genes) as being regulated significantly (P < 0.05) at one or more points; 50 were defined as regulated differentially with the false discovery rate at 10%. Significant changes in gene expression occurred in all categories as early as 2 hr post‐OBX, with the greatest number of differentially regulated genes at 16 and 48 hr. Hierarchical cluster analysis and correlation coefficients were used to identify similarities in patterns of gene expression changes within and across categories. Validation was carried out with SuperArray macroarrays and real‐time RT‐PCR. Our results confirmed the participation of many genes in known signaling pathways and identified changes in the expression of 42 genes not identified previously as participating in apoptosis and neurogenesis in the OE. Additionally, our analyses indicated the early involvement of genes regulating cytoskeletal reorganization and angiogenesis in the response to OBX. These studies are an important first step in defining early time‐dependent changes in gene expression after target ablation that lead to neurogenesis in the olfactory sensory epithelium. © 2005 Wiley‐Liss, Inc.
Tau is not normally degraded by the proteasome
Authors: Feuillette Sébastien , Blard Olivier , Lecourtois Magalie , Frébourg Thierry , Campion Dominique , Dumanchin Cécile
Published: March 2005

Tau‐positive inclusions in neurons are consistent neuropathologic features of the most common causes of dementias such Alzheimer's disease and frontotemporal dementia. Ubiquitinated tau‐positive inclusions have been reported in brains of Alzheimer's disease patients, but involvement of the ubiquitin‐dependent proteasomal system in tau degradation remains controversial. Before considering the tau degradation in pathologic conditions, it is important to determine whether or not endogenous tau is normally degraded by the proteasome pathway. We therefore investigated this question using two complementary approaches in vitro and in vivo. Firstly, SH‐SY5Y human neuroblastoma cells were treated with different proteasome inhibitors, MG132, lactacystin, and epoxomicin. Under these conditions, neither total nor phosphorylated endogenous tau protein levels were increased. Instead, an unexpected decrease of tau protein was observed. Secondly, we took advantage of a temperature‐sensitive mutant allele of the 20S proteasome in Drosophila. Genetic inactivation of the proteasome also resulted in a decrease of tau levels in Drosophila. These results obtained in vitro and in vivo demonstrate that endogenous tau is not normally degraded by the proteasome. © 2005 Wiley‐Liss, Inc.
Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers
Authors: Nagato Masako , Heike Toshio , Kato Takeo , Yamanaka Yasunari , Yoshimoto Momoko , Shimazaki Takuya , Okano Hideyuki , Nakahata Tatsutoshi
Published: March 2005

Neural stem cells (NSCs) with self‐renewal and multilineage differentiation properties can potentially repair degenerating or damaged neural tissue. Here, we have enriched NSCs from neurospheres, which make up a heterogeneous population, by fluorescence‐activated cell sorting (FACS) with antibodies against syndecan‐1, Notch‐1, and integrin‐β1, which were chosen as candidates for hematopoietic cell—or somatic stem cell—markers. Antigen‐positive cells readily initiated neurosphere formation, but cells lacking these markers did so less readily. Doubly positive cells expressing both syndecan‐1 and Notch‐1 underwent neurosphere formation more efficiently than did singly positive cells. The progeny of sorted cells could differentiate into neurons and glial cells both in vitro and in vivo. These antibodies were also useful for isolating cells from the murine embryonic day 14.5 brain that efficiently formed neurospheres. In contrast, there was no distinct difference in neurosphere formation efficiency between Hoechst 33342‐stained side population cells and main population cells, although the former are known to have a stem cell phenotype in various tissues. These results indicate the usefulness of syndecan‐1, Notch‐1, and integrin‐β1 as NSC markers. © 2005 Wiley‐Liss, Inc.
Glucose‐6‐phosphate dehydrogenase and NADPH‐consuming enzymes in the rat olfactory bulb
Authors: Biagiotti Enrica , Ferri Paola , Dringen Ralf , Del Grande Paolo , Ninfali Paolino
Published: March 2005

The resistance to oxidative stress is a multifactorial reaction involving the clustering of transcriptionally regulated genes. Because glucose‐6‐phosphate dehydrogenase (G6PD), the principal enzyme responsible for reducing power, is highly expressed in the olfactory bulb (OB), it is of interest to verify whether other enzymes utilizing NADPH are also highly expressed. The level and localization of G6PD‐ and NADPH‐consuming enzymes, such as NADPH‐cytochrome P450 oxidoreductase (P450R), glutathione reductase (GR), and NADPH‐diaphorase (NADPH‐d), were analyzed in the rat olfactory bulb (OB) by quantitative histochemistry and immunohistochemistry. The highest concentration of G6PD, P450R, and GR was observed in the olfactory nerve layer (ONL), suggesting a correlation in the expression of these enzymes at the gene level. Correlation in staining intensity between G6PD and NADPH‐d activities occurred only in part of the ONL, some glomeruli, and scattered periglomerular cells. This peculiar distribution of NADPH‐d could reflect a spatial patterning of the nose to bulb projections. Taken together, these results indicate that G6PD expression in the ONL could be related to the importance of generating a substantial supply of NADPH to sustain the detoxifying systems represented by GR and P450R reactions and, only in discrete zones, by NADPH‐d activity. © 2005 Wiley‐Liss, Inc.
Regulation of NT‐3 and BDNF levels in guinea pig auditory brain stem nuclei after unilateral cochlear ablation
Authors: Suneja Sanoj K. , Yan Leqin , Potashner Steven J.
Published: March 2005

Injury to areas of the central nervous system can alter neurotrophin levels, which may influence postlesion neuronal survival and plasticity. To determine if sensorineural hearing loss induces such changes, we used an enzyme‐linked immunosorbent assay (ELISA) to measure neurotrophin‐3 (NT‐3) and brain‐derived neurotrophic factor (BDNF) levels in adult guinea pig brain stem auditory nuclei 3–60 days after a unilateral cochlear ablation (UCA). After UCA, which destroyed the cochlea and cochlear nerve on one side, NT‐3 levels were usually depressed at 3 days by 22–44% but became elevated transiently at 7 days by 28–124%. BDNF levels were elevated transiently by 50% on the ablated side in the anteroventral (AVCN) and posteroventral (PVCN) cochlear nucleus at 3 days and may have signaled support for the survival of deafferented neurons. Coincident elevation at 3 and 7 days of BDNF or NT‐3 and phosphorylated extracellular signal‐regulated protein kinase 2 (ERK2‐P) suggested a relationship to stimulated signal transduction activity. Elevated neurotrophin levels may have contributed to synaptogenesis in the AVCN and the superior olive and to changes in the synaptic biochemistry in the auditory nuclei after UCA. In contrast, deficiencies or failure to elevate neurotrophin levels within several days of the UCA correlated with upregulation of phosphorylated stress‐activated protein kinase (SAPK‐P), suggesting a relationship with stress‐activated signal transduction and with the sparse degeneration of fibers observed in some of the auditory nuclei after UCA. © 2005 Wiley‐Liss, Inc.
Reduced CSF PLTP activity in Alzheimer's disease and other neurologic diseases; PLTP induces ApoE secretion in primary human astrocytes in vitro
Authors: Vuletic Simona , Peskind Elaine R. , Marcovina Santica M. , Quinn Joseph F. , Cheung Marian C. , Kennedy Hal , Kaye Jeffrey A. , Jin Lee‐Way , Albers John J.
Published: March 2005

Phospholipid transfer protein (PLTP) plays a pivotal role in cellular lipid efflux and modulation of lipoprotein metabolism. PLTP is distributed widely in the central nervous system (CNS), is synthesized by glia and neurons, and is active in cerebrospinal fluid (CSF). The aims of this study were to test the hypothesis that patients with Alzheimer's disease (AD) have altered PLTP‐mediated phospholipid transfer activity in CSF, and to examine the potential relationship between PLTP activity and apolipoprotein E (apoE) levels in CSF. We assessed PLTP activity and apoE concentration in CSF of patients with probable AD (n = 50), multiple sclerosis (MS; n = 9), other neurologic diseases (n = 21), and neurologically healthy controls (n = 40). PLTP activity in AD was reduced compared to that in controls (P < 0.001), with approximately half of the AD patients with PLTP activity values below all controls. Patients with MS had lower PLTP activity than AD patients (P < 0.001). PLTP activity was highly correlated with PLTP mass, as estimated by Western blot (r = 0.006; P < 0.01). CSF PLTP activity positively correlated with apoE concentration in AD (R = 0.435; P = 0.002) and controls (R = 0.456; P = 0.003). Anti‐apoE immunoaffinity chromatography and Western blot analyses indicated that some CSF PLTP is associated with apoE‐containing lipoproteins. Exogenous addition of recombinant PLTP to primary human astrocytes significantly increased apoE secretion to the conditioned medium. The findings of reduced PLTP activity in AD CSF, and the observation that PLTP can influence apoE secretion in astrocytes suggest a potential link between alterations in the brain lipid metabolism and AD pathogenesis. © 2005 Wiley‐Liss, Inc.
Drosophila melanogaster as a complementary system for studying HIV‐1‐related genes and proteins
Authors: Spresser Cole R. , Carlson Kimberly A.
Published: March 2005

Human immunodeficiency virus type 1 (HIV‐1) persists as a pandemic even though new information about the virus is being discovered on a daily basis. If the brain becomes infected, HIV‐1 encephalitis or HIV‐1‐associated dementia may develop. There is much to be learned about the modes of action and mechanisms of genes and proteins, and their interactions that underlie HIV‐1 infection. Drosophila melanogaster has been used successfully to study genes and proteins related to HIV‐1 infection, including but not limited to the disturbance of antimicrobial responses by viral protein U and the identification of D. melanogaster analogs to the serine palmitoyltransferase 5 and 6 proteins that play a role in activation of transcription by the HIV‐1 Tat protein in human cells. We believe that utilizing D. melanogaster as a complementary system for the study of genes and proteins related to HIV‐1 infection will provide useful information that will lead to new studies designed to enhance our understanding of the mechanistic roles of these molecules. In the present study, we focus on the utilization of D. melanogaster as a complementary system for studying HIV‐1 related genes and proteins, why this research should be extended, and why this complementary system is an important method for enhancing our understanding of the genetics involved in HIV‐1 infection. © 2005 Wiley‐Liss, Inc.
Selective caspase activation may contribute to neurological dysfunction after experimental spinal cord trauma
Authors: Knoblach S.M. , Huang X. , VanGelderen J. , Calva‐Cerqueira D. , Faden A.I.
Published: March 2005

Caspases are implicated in apoptotic cell death after spinal cord injury (SCI), but the relative contribution of these proteases to the secondary injury process has been only partially described. We examined the activation of caspases 1, 2, 3, 6, 8, and 9 from 1 hr to 7 days after moderate contusion injury induced by a weight‐drop method in the rat. Tissue homogenates from a 1‐cm segment of cord that contained the site of impact were processed by fluorometric enzymatic activity assays and/or immunoblotting methods. Caspases 3, 8, and 9 were activated from 1 to 72 hr after injury, whereas caspases 1, 2, and 6 were not. Double‐label immunohistochemistry utilizing antibodies for CNS cell‐type‐specific markers and active subunits of caspases 3, 8, or 9 showed that, at 4 and 72 hr after injury, these caspases were primarily activated in neurons and oligodendrocytes, rather than in astrocytes. Active caspase subunits were present in neurons within the necrotic lesion core at 4 hr after injury and in cells more than several segments away at 4 or 72 hr after injury. Intrathecal injection of the pan‐caspase inhibitor Boc‐Asp (OMe)‐fluoromethylketone (Boc‐d‐fmk) at 15 min after injury improved locomotor function 21 and 28 days later. Treatment with the selective caspase 3 inhibitor N‐benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp‐fluoromethyl ketone (z‐DEVD‐fmk) improved function at 21 days after injury. These data suggest that caspases 3, 8, and 9 may be differentially activated in white and gray matter after spinal cord trauma and that such activation may contribute to subsequent neurological dysfunction. © 2005 Wiley‐Liss, Inc.
Systematic administration of iptakalim, an ATP‐sensitive potassium channel opener, prevents rotenone‐induced motor and neurochemical alterations in rats
Authors: Yang Yong , Liu Xing , Long Yan , Wang Fang , Ding Jian‐Hua , Liu Su‐Yi , Sun Ye‐Hong , Yao Hong‐Hong , Wang Hai , Wu Jie , Hu Gang
Published: March 2005

Our previous studies revealed that iptakalim, a novel ATP‐sensitive potassium channel opener, has a significant neuroprotective function against ischemia in vivo or rotenone‐induced neurotoxicity in vitro. To investigate the potential pharmaceutical benefit of ATP‐sensitive potassium channel openers on neurodegenerative diseases, we studied the effects of iptakalim and diazoxide, a selective mitochondrial ATP‐sensitive potassium channel opener, on the rotenone‐induced nigrostriatal degeneration in rats. Iptakalim (1.5 mg/kg/day, orally) or diazoxide (1.5 mg/kg/day, orally) alone was administered to rats for 3 days, and then for 4 weeks was used daily with an injection of rotenone (2.5 mg/kg/day, subcutaneously) 1 hr later each time. The results showed that rotenone‐infused rats exhibited parkinsonian symptoms and had dopamine depletion in the striatum and substantia nigra. Pretreatment with iptakalim or diazoxide prevented rotenone‐induced catalepsy and the reduction of striatum dopamine contents. Moreover, iptakalim and diazoxide reduced the enzymatic activities and mRNA levels of inducible nitric oxide synthase elicited by chronic administration of rotenone. These neuroprotective effects of iptakalim and diazoxide were abolished by 5‐hydroxydecanoate, a selective mitochondrial ATP‐sensitive potassium channel blocker. In conclusion, our data suggested that mitochondrial ATP‐sensitive potassium channels might play a key role in preventing both parkinsonian symptoms and neurochemistry alterations induced by rotenone in rats. The selective activation of mitochondrial ATP‐sensitive potassium channels may provide a new therapeutic strategy for prevention and treatment of neurodegenerative disorders such as Parkinson's disease. © 2005 Wiley‐Liss, Inc.
Class groups of dihedral extensions
Authors: Lemmermeyer Franz
Published: April 2005

Let L/F be a dihedral extension of degree 2p, where p is an odd prime. Let K/F and k/F be subextensions of L/F with degrees p and 2, respectively. Then we will study relations between the p‐ranks of the class groups Cl(K) and Cl(k). (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Minimal regularity of the solution of some boundary value problems of Signorini's type in polygonal domains
Authors: Mercier Denis , Nicaise Serge
Published: April 2005

We study the regularity in Sobolev spaces of the solution of transmission problems in a polygonal domain of the plane, with unilateral boundary conditions of Signorini's type in a part of the boundary and Dirichlet or Neumann boundary conditions on the remainder part. We use a penalization method combined with an appropriated lifting argument to get uniform estimates of the approximated solutions in order to obtain some minimal regularity results for the exact solution. The same method allows us to consider problems with thin obstacles. It can be easily extended to 3D problems. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Bounding Poincaré‐Hopf indices and Milnor numbers
Authors: Soares Marcio G.
Published: April 2005

We use Mather's finite determinacy theory and Baum‐Bott's theorem to give sharp bounds for the Poincaré‐Hopf index of a germ of homolorphic vector field with an isolated zero. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Distal enhancer of the mouse FGF‐4 gene and its human counterpart exhibit differential activity: Critical role of a GT box
Authors: Boer Brian , Luster Troy A. , Bernadt Cory , Rizzino Angie
Published: April 2005

Previous studies have shown that there is a strict requirement for fibroblast growth factor‐4 (FGF‐4) during mammalian embryogenesis, and that FGF‐4 expression in embryonic stem (ES) cells and embryonal carcinoma (EC) cells are controlled by a powerful downstream distal enhancer. More recently, mouse ES cells were shown to express significantly more FGF‐4 mRNA than human ES cells. In the work reported here, we demonstrate that mouse EC cells also express far more FGF‐4 mRNA than human EC cells. Using a panel of FGF‐4 promoter/reporter gene constructs, we demonstrate that the enhancer of the mouse FGF‐4 gene is approximately tenfold more active than its human counterpart. Moreover, we demonstrate that the critical difference between the mouse and the human FGF‐4 enhancer is a 4 bp difference in the sequence of an essential GT box. Importantly, we demonstrate that changing 4 bp in the human enhancer to match the sequence of the mouse GT box elevates the activity of the human FGF‐4 enhancer to the same level as that of the mouse enhancer. We extended these studies by examining the roles of Sp1 and Sp3 in FGF‐4 expression. Although we demonstrate that Sp3, but not Sp1, can activate the FGF‐4 promoter when artificially tethered to the FGF‐4 enhancer, we show that Sp3 is not essential for expression of FGF‐4 mRNA in mouse ES cells. Finally, our studies with human EC cells suggest that the factor responsible for mediating the effect of the mouse GT box is unlikely to be Sp1 or Sp3, and this factor is either not expressed in human EC cells or it is not sufficiently active in these cells. Mol. Reprod. Dev. © 2005 Wiley‐Liss, Inc.
Unequal segregation of parental chromosomes in embryonic stem cell hybrids
Authors: Matveeva Natalia M. , Pristyazhnyuk Inna E. , Temirova Symbat A. , Menzorov Alexey G. , Vasilkova Anna , Shilov Alexander G. , Smith Austin , Serov Oleg L.
Published: April 2005

Chromosome segregation was studied in 14 intra‐ and 20 inter‐specific hybrid clones generated by fusion of Mus musculus embryonic stem (ES) cells with fibroblasts or splenocytes of DD/c mice or Mus caroli. As a control for in vitro evolution of tetraploid karyotype we used a set of hybrid clones obtained by fusion of ES cells (D3) with ES cells (TgTP6.3). Identification of the parental chromosomes in the clones was performed by microsatellite analysis and in situ hybridization with labeled species‐specific probes. Both analyses have revealed three types of clones: (i) stable tetraploid, observed only for ES × ES cell hybrids; (ii) bilateral loss of chromosomes of both ES and somatic partners; (iii) unilateral segregation of chromosomes of the somatic partner. Observed unilateral segregation was extensive in ES–splenocyte cell hybrids, but lower in ES–fibroblast hybrid clones. Developmental state of the somatic partner is presumably responsible for directional chromosome loss. Nonrandom segregation implies that initial differences in the parental homologous chromosomes were not immediately equalized implying at least transient persistence of the differentiated epigenotype. Mol. Reprod. Dev. © 2005 Wiley‐Liss, Inc.
PLAC1 expression increases during trophoblast differentiation: Evidence for regulatory interactions with the fibroblast growth factor‐7 (FGF‐7) axis
Authors: Massabbal Eltayab , Parveen Shanaz , Weisoly D.L. , Nelson D. Michael , Smith S.D. , Fant Michael
Published: April 2005

PLAC1 is a recently described, trophoblast‐specific gene that localizes to a region of the X‐chromosome important in placental development. Immunohistochemical analysis demonstrated that PLAC1 polypeptide localizes to the differentiated syncytiotrophoblast throughout gestation (8–41 weeks) as well as a small population of villous cytotrophoblasts. Consistent with these observations, quantitative RT‐PCR demonstrated that PLAC1 mRNA increases more than 300‐fold during cytotrophoblast differentiation in culture to form syncytiotrophoblasts. Agents known to be relevant to trophoblast differentiation were then tested for the ability to influence PLAC1 expression. Fibroblast growth factor‐7 (FGF‐7), also known as keratinocyte growth factor (KGF), stimulated PLAC1 mRNA expression approximately two‐fold in the BeWob30 trophoblast cell line. FGF‐7 stimulation was significantly inhibited by PD‐98059 and wortmannin suggesting mediation via MAP kinase and PI‐3 kinase‐dependent signaling pathways. Interestingly, epidermal growth factor (EGF) treatment of trophoblasts had no effect on PLAC1 expression alone, but potentiated the effect of FGF‐7, suggesting the presence of a regulatory interaction of the two growth factors. FGF‐7 and its receptor, FGFR‐2b, exhibited spatial overlap with PLAC1 suggesting these regulatory interactions are physiologically relevant during gestation. These data demonstrate PLAC1 expression is upregulated during trophoblast differentiation, localizing primarily to the differentiated syncytiotrophoblast. Furthermore PLAC1 expression is specifically regulated by peptide growth factors relevant to trophoblast differentiation. Mol. Reprod. Dev. © 2005 Wiley‐Liss, Inc.
Actions of progesterone on uterine immunosuppression and endometrial gland development in the uterine gland knockout (UGKO) ewe
Authors: Padua Maria B. , Tekin Şaban , Spencer Thomas E. , Hansen Peter J.
Published: April 2005

In ewes, the uterine gland knockout (UGKO) phenotype is caused by neonatal exposure to norgestomet to arrest uterine gland development and produce an adult which has a uterus characterized by the lack of endometrial glands. Since endometrial glands in the sheep produce the lymphocyte‐inhibitory protein, ovine uterine serpin (OvUS), an experiment was conducted with ewes of the UGKO phenotype to evaluate whether the inhibitory actions of progesterone on tissue rejection responses in utero are dependent upon the presence of endometrial glands. Control and UGKO ewes were ovariectomized and subsequently treated with either 100 mg/day progesterone or corn oil vehicle for 30 days. An autograft and allograft of skin were then placed in each uterine lumen and treatments were continued for an additional 30 days before grafts were examined for survival. All autografts survived and had a healthy appearance after histological analysis. Allografts were generally rejected in ewes treated with vehicle but were present for hormone‐treated ewes, regardless of uterine phenotype. Analysis of the histoarchitecture and protein synthetic capacity of the uterus revealed that progesterone induced differentiation of endometrial glands and synthesis and secretion of OvUS in UGKO ewes. The UGKO ewes had reduced density of CD45R+ lymphocytes in the endometrial epithelium and there was a tendency for progesterone to reduce this effect in luminal epithelium. Taken together, results confirm the actions of progesterone to inhibit graft rejection response in utero. Responses of UGKO ewes to progesterone indicate that the hormone can induce de novo development and differentiation of endometrial glands, at least when skin grafts are in the uterus. Mol. Reprod. Dev. © 2005 Wiley‐Liss, Inc.
Developmental capability of denuded bovine oocyte in a Co‐culture system with intact cumulus‐oocyte complexes: Role of cumulus cells, cyclic adenosine 3′,5′‐monophosphate, and glutathione
Authors: Luciano Alberto M. , Lodde Valentina , Beretta Matteo S. , Colleoni Silvia , Lauria Antonio , Modina Silvia
Published: April 2005

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte‐sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co‐cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus‐oocyte complexes (COCs) in co‐culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3′–5′ monophosphate (cAMP) and glutathione (GSH) during FSH‐stimulated maturation of denuded oocyte co‐cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co‐presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co‐cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete. Mol. Reprod. Dev. © 2005 Wiley‐Liss, Inc.
Ophthalmoplegia due to mitochondrial DNA disease: The need for genetic diagnosis
Authors: Schaefer Andrew M. , Blakely Emma L. , Griffiths Philip G. , Turnbull Douglass M. , Taylor Robert W.
Published: March 2005

We describe a patient with chronic progressive external ophthalmoplegia (CPEO) who underwent muscle biopsy for suspected mitochondrial disease. In spite of normal histocytochemical cytochrome c oxidase (COX) activity and respiratory chain enzyme measurements in muscle, subsequent molecular genetic analysis revealed the presence of a single, large‐scale deletion of mitochondrial DNA (mtDNA). The case serves to illustrate the importance of pursuing the proposed mitochondrial genetic abnormality, even in patients with normal biopsy findings. Muscle Nerve, 2005
Fluctuations in motor output during steady contractions are weakly related across contraction types and between hands
Authors: Shinohara Minoru , Keenan Kevin G. , Enoka Roger M.
Published: March 2005

The presence of differences in motor unit activity across contraction types and between hands suggests that the magnitude of fluctuations in motor output is only weakly related when these conditions are compared. Twenty right‐handed young (24.1 ± 5.3 years) and old (72.5 ± 4.9 years) adults performed three levels (≤40% of maximal force) of isometric and anisometric contractions with the first dorsal interosseous muscle of each hand. The fluctuations in motor output were quantified by the coefficient of variation for force during isometric contractions, and as the standard deviations of acceleration and position during anisometric contractions. There was no effect of age on fluctuations in motor output. The magnitude of the fluctuations in motor output was weakly related across contraction types (r2 < 0.325) and between hands (r2 < 0.262). Furthermore, the standard deviations of acceleration and position during shortening and lengthening contractions were largely unrelated to one another. Because the activity in a motor unit population differed across contraction types and hands during steady contractions, the central nervous system likely employed distinct strategies to accomplish these different tasks. Muscle Nerve, 2005
Influence of knee joint angle on muscle properties of paralyzed and nonparalyzed human knee extensors
Authors: Gerrits Karin H. , Maganaris Constantinos N. , Reeves Neil D. , Sargeant Anthony J. , Jones David A. , de Haan Arnold
Published: March 2005

Muscles of individuals with a spinal cord injury (SCI) exhibit an unexpected leftward shift in the force (torque)–frequency relationship. We investigated whether differences in torque–angle relationships between SCI and able‐bodied control muscles could explain this shift. Electrically stimulated knee‐extensor contractions were obtained at knee flexion angles of between 30° and 90°. Torque–frequency relationships were obtained at 30°, 90°, and optimum angle. Optimum angle was not different between groups but SCI‐normalized torques were lower at the extreme angles. At all angles, SCI muscles produced higher relative torques at low stimulation frequencies. Thus, there was no evidence of a consistent change in the length of paralyzed SCI muscles, and the anomalous leftward shift in the torque–frequency relationship was not the result of testing the muscle at a relatively long length. The results provide valuable information about muscle changes occurring in various neurological disorders. Muscle Nerve, 2005