bluuebunny/crossref_metadata_2025 huggingface dataset:
Microarray analyses identify molecular biomarkers of Atlantic salmon macrophage and hematopoietic kidney response toPiscirickettsia salmonisinfection
Authors: Rise Matthew L. , Jones Simon R. M. , Brown Gordon D. , von Schalburg Kristian R. , Davidson William S. , Koop Ben F.
Published: September 2004

Piscirickettsia salmonis is the intracellular bacterium that causes salmonid rickettsial septicemia, an infectious disease that kills millions of farmed fish each year. The mechanisms used by P. salmonis to survive and replicate within host cells are not known. Piscirickettsiosis causes severe necrosis of hematopoietic kidney. Microarray-based experiments with QPCR validation were used to identify Atlantic salmon macrophage and hematopoietic kidney genes differentially transcribed in response to P. salmonis infection. Infections were confirmed by microscopy and RT-PCR with pathogen-specific primers. In infected salmon macrophages, 71 different transcripts were upregulated and 31 different transcripts were downregulated. In infected hematopoietic kidney, 30 different transcripts were upregulated and 39 different transcripts were downregulated. Ten antioxidant genes, including glutathione S-transferase, glutathione reductase, glutathione peroxidase, and cytochrome b558 α- and β-subunits, were upregulated in infected macrophages but not in infected hematopoietic kidney. Changes in redox status of infected macrophages may allow these cells to tolerate P. salmonis infection, raising the possibility that treatment with antioxidants may reduce hematopoietic tissue damage caused by this rickettsial infection. The downregulation of transcripts involved in adaptive immune responses (e.g., T cell receptor α-chain and C-C chemokine receptor 7) in infected hematopoietic kidney but not in infected macrophages may contribute to infection-induced kidney tissue damage. Molecular biomarkers of P. salmonis infection, characterized by immune-relevant functional annotations and high fold differences in expression between infected and noninfected samples, may aid in the development of anti-piscirickettsial vaccines and therapeutics.
DNA microarray analysis of gene expression in alveolar epithelial cells in response to TNFα, LPS, and cyclic stretch
Authors: dos Santos C.C. , Han B. , Andrade C.F. , Bai X. , Uhlig S. , Hubmayr R. , Tsang M. , Lodyga M. , Keshavjee S. , Slutsky A.S. , Liu M.
Published: September 2004

Recent evidence suggests that alveolar epithelial cells (AECs) may contribute to the development, propagation, and resolution of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Proinflammatory cytokines, pathogen products, and injurious mechanical ventilation are important contributors of excessive inflammatory responses in the lung. In the present study, we used cDNA microarrays to define the gene expression patterns of A549 cells (an AEC line) in the early stages of three models of pulmonary parenchymal cell activation: cells treated with tumor necrosis factor-α (TNFα) (20 ng/ml), lipopolysaccharide (LPS, 1 μg/ml), or cyclic stretch (20% elongation) for either 1 h or 4 h. Differential gene expression profiles were determined by gene array analysis. TNFα induced an inflammatory response pattern, including induction of genes for chemokines, inflammatory mediators, and cell surface membrane proteins. TNFα also increased genes related to pro- and anti-apoptotic proteins, signal transduction proteins, and transcriptional factors. TNFα further induced a group of genes that may form a negative feedback loop to silence the NFκB pathway. Stimulation of AECs with mechanical stretch changed cell morphology and activated Src protein tyrosine kinase. The combination of TNFα plus stretch enhanced or attenuated expression of multiple genes. LPS decreased microfilament polymerization but had less impact on NFκB translocation and gene expression. Results from this study indicate that AECs can tailor their response to different stimuli or/and combination of stimuli and subsequently play an important role in acute inflammatory responses in the lung.
Functional defect of regulatory CD4+CD25+ T cells in the thymus of patients with autoimmune myasthenia gravis
Authors: Balandina Anna , Lécart Sandrine , Dartevelle Philippe , Saoudi Abdelhadi , Berrih-Aknin Sonia
Published: September 2004

Thymus-derived CD4+CD25+ regulatory T (Treg) cells are essential for the maintenance of immunologic self-tolerance. Despite their critical role in the active suppression of experimental autoimmune disorders, little is known about their involvement in human autoimmune diseases. Myasthenia gravis (MG) is a CD4+ T cell–dependent autoimmune disease and the thymus is assumed to be the initiation site. To identify possible defects in the Treg cells in MG, we analyzed CD4+CD25+ cells in thymi from patients with MG compared to those from healthy subjects. We found a normal CD4+CD25+ number but a severe functional defect in their regulatory activity together with a decreased expression of the transcription factor, Foxp3, which is essential for T-cell regulatory function. The phenotypic analysis of CD4+CD25+ thymocytes revealed an increased number of activated effector cells with strong Fas expression in patients with MG. However, whatever their level of Fas, CD4+CD25+ thymocytes from patients with MG remained unable to suppress the proliferation of responding cells, indicating that the impaired Treg cell function is not due to contamination by activated effector T cells. These data are the first to demonstrate a severe functional impairment of thymic Treg cells in MG, which could contribute to the onset of this autoimmune disease.
Imatinib mesylate (STI-571) enhances antigen-presenting cell function and overcomes tumor-induced CD4+ T-cell tolerance
Authors: Wang Hongwei , Cheng Fengdong , Cuenca Alex , Horna Pedro , Zheng Zheng , Bhalla Kapil , Sotomayor Eduardo M.
Published: September 2004

Tumor antigen–specific T-cell tolerance imposes a significant barrier to the development of effective therapeutic cancer vaccines. Bone marrow–derived antigen-presenting cells (APCs) are critical in the induction of this unresponsive state. Here we show that in vitro treatment of APCs with the tyrosine kinase inhibitor, imatinib mesylate (STI-571), enhances the activation of naive antigen-specific T cells and restores the responsiveness of tolerant T cells from tumor-bearing hosts. Furthermore, in vivo treatment with STI-571 not only prevented the induction of tolerance in tumor-specific CD4+ T cells, preserving their responsiveness to a subsequent immunization, but also resulted in enhanced vaccine efficacy. These findings demonstrate that tolerance to tumor antigens is not an insurmountable obstacle and points to modulation of APC function as a promising strategy in the immunotherapy of cancer.
Identification of cooperative genes for NUP98-HOXA9 in myeloid leukemogenesis using a mouse model
Authors: Iwasaki Masayuki , Kuwata Takeshi , Yamazaki Yukari , Jenkins Nancy A. , Copeland Neal G. , Osato Motomi , Ito Yoshiaki , Kroon Evert , Sauvageau Guy , Nakamura Takuro
Published: September 2004

The chromosomal translocation t(7; 11)(p15;p15), observed in human myeloid leukemia, results in a NUP98 and HOXA9 gene fusion. We generated a transgenic mouse line that specifically expressed the chimeric NUP98-HOXA9 gene in the myeloid lineage. While only 20% of the transgenic mice progressed to leukemia after a latency period, myeloid progenitor cells from nonleukemic transgenic mice still exhibited increased proliferative potential. This suggested that the NUP98-HOXA9 fusion induced a preleukemic phase, and other factors were required for complete leukemogenesis. NUP98-HOXA9 expression promoted the onset of retrovirus-induced BXH2 myeloid leukemia. This phenomenon was used to identify cooperative disease genes as common integration sites (CISs). Meis1, a known HOX cofactor, was identified as a CIS with a higher integration frequency in transgenic than in wild-type BXH2 mice. By the same means we identified further 4 candidate cooperative genes, Dnalc4, Fcgr2b, Fcrl, and Con1. These genes cooperated with NUP98-HOXA9 in transforming NIH 3T3 cells. The system described here is a powerful tool to identify cooperative oncogenes and will assist in the clarification of the multistep process of carcinogenesis.
Influence of CD33 expression levels and ITIM-dependent internalization on gemtuzumab ozogamicin–induced cytotoxicity
Authors: Walter Roland B. , Raden Brian W. , Kamikura Darren M. , Cooper Jonathan A. , Bernstein Irwin D.
Published: September 2004

Gemtuzumab ozogamicin (GO; Mylotarg), a novel immunoconjugate used for treatment of acute myeloid leukemia (AML), contains the humanized anti-CD33 antibody (hP67.6) as a carrier to facilitate cellular uptake of the toxic calicheamicin-γ1 derivative. By use of lentivirus-mediated gene transfer to manipulate CD33 expression in myeloid cell lines that normally lack CD33 (murine 32D cells) or have very low levels of CD33 (human OCI-AML3 and KG-1a cells), we here show a quantitative relationship between CD33 expression and GO-induced cytotoxicity. The CD33 cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) control internalization of antibody bound to CD33. Disruption of the ITIMs by introduction of point mutations not only prevented effective internalization of antibody-bound CD33 but also significantly reduced GO-induced cytotoxicity. Together, our data imply a pivotal role of both the number of CD33 molecules expressed on the cell surface and the amount of internalization of CD33 following antibody binding for GO-induced cytotoxicity and suggest novel therapeutic approaches for improvement of clinical outcome of patients treated with GO.
Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque
Authors: Ferrero Enza , Orciani Monia , Vacca Paola , Ortolan Erika , Crovella Sergio , Titti Fausto , Saccucci Franca , Malavasi Fabio
Published: September 2004

Background

The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38.

Results

A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of ~42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop.

Conclusion

This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.
Development of a method for screening short-lived proteins using green fluorescent protein
Authors: Jiang Xin , Coffino Philip , Li Xianqiang
Published: September 2004

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.
Randomized Phase III Trial of Pegylated Liposomal Doxorubicin Versus Vinorelbine or Mitomycin C Plus Vinblastine in Women With Taxane-Refractory Advanced Breast Cancer
Authors: Keller Alan M. , Mennel Robert G. , Georgoulias Vassilis A. , Nabholtz Jean-Marc , Erazo Aura , Lluch Anna , Vogel Charles L. , Kaufmann Manfred , von Minckwitz Gunther , Henderson I. Craig , Mellars Lillian , Alland Leila , Tendler Craig
Published: September 2004

Purpose

To compare the efficacy of pegylated liposomal doxorubicin (PLD) with that of a common salvage regimen (comparator) in patients with taxane-refractory advanced breast cancer.

Patients and Methods

Following failure of a first- or second-line taxane-containing regimen for metastatic disease, 301 women were randomly assigned to receive PLD (50 mg/m2 every 28 days); or comparator-vinorelbine (30 mg/m2 weekly) or mitomycin C (10 mg/m2 day 1 and every 28 days) plus vinblastine (5 mg/m2 day 1, day 14, day 28, and day 42) every 6 to 8 weeks. Patients were stratified before random assignment based on number of previous chemotherapy regimens for metastatic disease and presence of bone metastases only.

Results

Progression-free survival (PFS) and overall survival (OS) were similar for PLD and comparator (PFS: hazard ratio [HR], 1.26; 95% CI, 0.98 to 1.62; P = .11; median, 2.9 months [PLD] and 2.5 months [comparator]; OS: HR, 1.05; 95% CI, 0.82 to 1.33; P = .71; median, 11.0 months [PLD] and 9.0 months [comparator]). In anthracycline-naïve patients, PFS was somewhat longer with PLD, relative to the comparator (n = 44; median PFS, 5.8 v 2.1 months; HR, 2.40; 95% CI, 1.16 to 4.95; P = .01). Most frequently reported adverse events were nausea (23% to 31%), vomiting (17% to 20%), and fatigue (9% to 20%) and were similar among treatment groups. PLD-treated patients experienced more palmar-plantar erythrodysesthesia (37%; 18% grade 3, 1 patient grade 4) and stomatitis (22%; 5% grades 3/4). Neuropathy (11%), constipation (16%), and neutropenia (14%) were more common with vinorelbine. Alopecia was low in both the PLD and vinorelbine groups (3% and 5%).

Conclusion

PLD has efficacy comparable to that of common salvage regimens in patients with taxane-refractory metastatic breast cancer, thereby representing a useful therapeutic option.
Imitative Games by 9‐, 14‐, and 18‐Month‐Old Infants
Authors: Agnetta Bryan , Rochat Philippe
Published: September 2004

Two experiments used a mutual imitation paradigm to assess 9‐, 14‐, and 18‐month‐old infants' developing understanding of intentions in others. In the first study, 1 experimenter imitated the infants' actions, and another experimenter performed contingent but different actions on an identical toy. From 9 months of age, infants show discrimination between the mimicking and the contingent experimenter. In a second study, same‐age groups of infants faced either an experimenter mimicking their actions on an identical object or the object mimicking them independently of any manual contact by the experimenter. Only 14‐ and 18‐month‐olds showed discrimination between the 2 conditions, this discrimination correlating with infants' relative ability to follow gaze and points in triadic exchanges. These results are interpreted as demonstrating important developmental changes between 9 and 14 months in the construal of others as intentional.
Physiological and Emotional Reactivity to Learning and Frustration
Authors: Lewis Michael , Hitchcock Daniel F. A. , Sullivan Margaret Wolan
Published: September 2004

This study examined the behavioral (arm, facial) and autonomic (heart rate, respiratory sinus arrhythmia [RSA], and adrenocortical axis) reactivity of 56 4‐month‐old infants in response to contingency learning and extinction‐induced frustration. During learning, infants displayed increases in operant arm response and positive emotional expressions. Changes in average RSA(VNA) paralleled the observed changes in facial expressions in general and maintained an inverse relation with heart rate throughout most of the session. When frustrated by extinction, infants displayed increases in negative expressions, heart rate, and a brief increase in RSA(VNA) followed by a significant decrease. No significant changes were observed for Cortisol. These behavioral and facial responses are consistent with earlier work. The physiological changes, along with the facial expressions and instrumental responses, indicate that the autonomic nervous system functions as a coordinated affect system by 4 months of age.
Progesterone inhibits protein kinase A (PKA) inXenopusoocytes: demonstration of endogenous PKA activities using an expressed substrate
Authors: Wang Jing , Liu X. Johné
Published: September 2004

3′-5′ cyclic adenosine monophosphate (cAMP)-dependent protein kinase, PKA, is thought to be a key enzyme that controls prophase arrest in vertebrate oocytes. It has long been established that overexpression of the catalytic subunit of PKA inhibits hormone-induced frog oocyte maturation whereas overexpression of the regulatory subunits induces hormone-independent oocyte maturation. However, the activities of endogenous oocyte PKA, or its regulation by the maturation-inducing hormone progesterone, have never been directly demonstrated in frog oocytes. We have developed a novel expressed substrate for PKA in live oocytes by constructing a fusion protein containing an N-terminal myristylation sequence (derived from the Src tyrosine kinase) followed by an antigenic epitope tag and a substrate motif (the C-terminal cytoplasmic domain of β2 adrenergic receptor). Following mRNA injection, the phosphorylation status of the substrate was determined by two-dimensional electrophoresis followed by epitope immunoblotting, or alternatively by SDS-PAGE followed by immunoblotting using antibodies specifically recognizing the PKA-phosphorylated form of the substrate. In prophase oocytes, the expressed protein, myr-HA-β2AR-C, was fully phosphorylated on a single PKA site (Ser346 of human β2 adrenergic receptor). Within one hour of the addition of progesterone, the PKA site became mostly dephosphorylated. No re-phosphorylation of the PKA site, and therefore no reactivation of PKA, was observed throughout the entire maturation process. To demonstrate the generality of this PKA substrate, we analyzed its phosphorylation status in COS-7 cells following transfection. We show that dibutyryl cAMP rapidly stimulates phosphorylation of the PKA site. These results represent the first biochemical demonstration of regulation of endogenous Xenopus oocyte PKA by progesterone. Furthermore, myr-HA-β2AR-C should be widely adaptable as an in vivo PKA activity indicator.
N-Glycans, not the GPI anchor, mediate the apical targeting of a naturally glycosylated, GPI-anchored protein in polarised epithelial cells
Authors: Pang Susan , Urquhart Paula , Hooper Nigel M.
Published: September 2004

The glycosyl-phosphatidylinositol (GPI) anchor mediates the apical sorting of proteins in polarised epithelial cells through its interaction with lipid rafts. Here we investigated the signals required for the apical targeting of the naturally N-glycosylated and GPI-anchored membrane dipeptidase by selective point mutation to remove the GPI anchor addition signal or the sites for N-linked glycosylation, or both. Activity assays, immunoblotting and immunofluorescence microscopy revealed that the constructs lacking the GPI anchor were secreted from Madin-Darby canine kidney (MDCK) cells, whereas those retaining the GPI anchor were attached at the cell surface, irrespective of the glycosylation status. Wild-type membrane dipeptidase was expressed preferentially on the apical surface of both MDCK and CaCo-2 cells. By contrast, the GPI-anchored construct lacking the N-glycans was targeted preferentially to the basolateral surface of both cell types. In constructs lacking the GPI anchor, the N-glycans also targeted the protein to the apical surface. Both the apically targeted, glycosylated and the basolaterally targeted, unglycosylated GPI-anchored forms of the protein were located in detergent-insoluble lipid rafts. These data indicate that it is the N-glycans, not the association of the GPI anchor with lipid rafts, which determine apical targeting of an endogenously N-glycosylated, GPI-anchored protein in polarised epithelial cells.
Phosphorylation by cAMP-dependent protein kinase is essential for synapsin-induced enhancement of neurotransmitter release in invertebrate neurons
Authors: Fiumara Ferdinando , Giovedì Silvia , Menegon Andrea , Milanese Chiara , Merlo Daniela , Montarolo Pier Giorgio , Valtorta Flavia , Benfenati Fabio , Ghirardi Mirella
Published: September 2004

Synapsins are synaptic vesicle-associated phosphoproteins involved in the regulation of neurotransmitter release and synapse formation; they are substrates for multiple protein kinases that phosphorylate them on distinct sites. We have previously found that injection of synapsin into Helix snail neurons cultured under low-release conditions increases the efficiency of neurotransmitter release. In order to investigate the role of phosphorylation in this modulatory action of synapsins, we examined the substrate properties of the snail synapsin orthologue recently cloned in Aplysia (apSyn) for various protein kinases and compared the effects of the intracellular injection of wild-type apSyn with those of its phosphorylation site mutants. ApSyn was found to be an excellent in vitro substrate for cAMP-dependent protein kinase, which phosphorylated it at high stoichiometry on a single site (Ser-9) in the highly conserved domain A, unlike the other kinases reported to phosphorylate mammalian synapsins, which phosphorylated apSyn to a much lesser extent. The functional effect of apSyn phosphorylation by cAMP-dependent protein kinase on neurotransmitter release was studied by injecting wild-type or Ser-9 mutated apSyn into the soma of Helix serotonergic C1 neurons cultured under low-release conditions, i.e. in contact with the non-physiological target neuron C3. In this model of impaired neurotransmitter release, the injection of wild-type apSyn induced a significant enhancement of release. This enhancement was virtually absent after injection of the non-phosphorylatable mutant (Ser-9→Ala), but it was maintained after injection of the pseudophosphorylated mutant (Ser-9→Asp). These functional effects of apSyn injection were paralleled by marked ultrastructural changes in the C1 neuron, with the formation of extensive interdigitations of neurite-like processes containing an increased complement of C1 dense core vesicles at the sites of cell-to-cell contact. This structural rearrangement was virtually absent in mock-injected C1 neurons or after injection of the non-phosphorylatable apSyn mutant. These data indicate that phosphorylation of synapsin domain A is essential for the synapsin-induced enhancement of neurotransmitter release and suggest that endogenous kinases phosphorylating this domain play a central role in the regulation of the efficiency of the exocytotic machinery.
Maternal UNC-45 is involved in cytokinesis and colocalizes with non-muscle myosin in the early Caenorhabditis elegans embryo
Authors: Kachur Torah , Ao Wanyuan , Berger Jeffrey , Pilgrim Dave
Published: September 2004

The Caenorhabditis elegans UNC-45 protein contains tetratricopeptide repeats and a domain with similarity to fungal proteins, and it differentially colocalizes with myosin heavy chain B in the body wall muscles of adult worms. Although it is essential for normal myosin filament assembly in body wall muscle development, strong mutants show a previously unexplained maternal effect. We show here that the UNC-45 protein is maternally contributed and is present in all cells of the early embryo whereas zygotic UNC-45 expression is only detected in the developing muscle cells. Embryos produced from adults with reduced germline expression of UNC-45 exhibit cytokinesis defects suggesting that UNC-45 has a novel role in the early embryo in addition to muscle development. Yeast two-hybrid screens show that UNC-45 can directly interact with NMY-2, a non-muscle type II myosin, and UNC-45 and NMY-2 colocalize at cell boundaries in early embryos. Localization of UNC-45 at these boundaries is dependent upon the presence of NMY-2. Our results suggest that UNC-45 interacts with more than one type of myosin and functions in the embryo to regulate cytoplasmic myosin assembly and/or stability during cytokinesis.
Characterisation of Cdc25B localisation and nuclear export during the cell cycle and in response to stress
Authors: Lindqvist Arne , Källström Helena , Karlsson Rosenthal Christina
Published: September 2004

Cdc25 phosphatases are essential regulators of the cell cycle. In mammalian cells, the Cdc25B isoform activates cyclin A- and cyclin B1-containing complexes and is necessary for entry into mitosis. In this report, we characterise the subcellular localisation of Cdc25B by immunofluorescence in combination with RNA interference to identify specific antibody staining. We find that endogenous Cdc25B is mainly nuclear, but a fraction resides in the cytoplasm during the G2 phase of the cell cycle. Cdc25B starts to appear in S-phase cells and accumulates until prophase, after which the protein disappears. We characterise a nuclear export sequence in the N-terminus of Cdc25B (amino acids 54-67) that, when mutated, greatly reduces the ability of Cdc25B to shuttle in a fluorescence loss in photobleaching assay. Mutation of the nuclear export sequence makes Cdc25B less efficient in inducing mitosis, suggesting that an important mitotic function of Cdc25B occurs in the cytoplasm. Furthermore, we find that when cells are exposed to cycloheximide or ultraviolet irradiation, Cdc25B partially translocates to the cytoplasm. The dependence of this translocation event on a functional nuclear export sequence, an intact serine 323 residue (a 14-3-3 binding site) and p38 mitogen-activated protein kinase activity indicates that the p38 pathway regulates Cdc25B localisation in different situations of cellular stress.
Cathepsin D released by lactating rat mammary epithelial cells is involved in prolactin cleavage under physiological conditions
Authors: Lkhider Mustapha , Castino Roberta , Bouguyon Edwige , Isidoro Ciro , Ollivier-Bousquet Michèle
Published: September 2004

The 16 kDa prolactin fragment arises from partial proteolysis of the native 23 kDa prolactin pituitary hormone. The mammary gland has been involved in this processing, although it has not been clarified whether it occurs in stroma or epithelial cells or extracellularly. Also, the processing enzyme has not been defined yet. Here we show that the incubation medium of stroma-deprived mammary acini from lactating rat contains an enzymatic activity able to cleave, in a temperature- and time-dependent fashion, the 23 kDa prolactin to generate a 16 kDa prolactin detectable under reducing conditions. This cleavage was not impaired in the presence of hirudin, a thrombin inhibitor, but strongly weakened in the presence of pepstatin A, a cathepsin D inhibitor. Cathepsin D immuno-depletion abolished the capability of acini-conditioned medium to cleave the 23 kDa prolactin. Brefeldin A treatment of acini, a condition that largely abolished the apical secretion of milk proteins, did not impair the secretion of the enzymatically active single chain of cathepsin D. These results show that mature cathepsin D from endosomes or lysosomes is released, likely at the baso-lateral site of mammary epithelial cells, and that a cathepsin D-dependent activity is required to effect, under physiological conditions, the cleavage of 23 kDa prolactin in the extracellular medium. This is the first report demonstrating that cathepsin D can perform a limited proteolysis of a substrate at physiological pH outside the cell.
Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression
Authors: Guillemot Laurent , Hammar Eva , Kaister Christian , Ritz Jorge , Caille Dorothée , Jond Lionel , Bauer Christoph , Meda Paolo , Citi Sandra
Published: September 2004

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (–/–) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence. In hetero- and homozygous (+/– and –/–) embryoid bodies, immunoblotting revealed a Triton-soluble, truncated form of cingulin, increased levels of the tight junction proteins ZO-2, occludin, claudin-6 and Lfc, and decreased levels of ZO-1. The +/– and –/– embryoid bodies contained epithelial cells with normal tight junctions, as determined by freeze-fracture and transmission electron microscopy, and a biotin permeability assay. The localization of ZO-1, occludin and claudin-6 appeared normal in mutant epithelial cells, indicating that cingulin is not required for their junctional recruitment. Real-time quantitative reverse-transcription PCR (real-time qRT-PCR) showed that differentiation of embryonic stem cells into embryoid bodies was associated with up-regulation of mRNAs for several tight junction proteins. Microarray analysis and real-time qRT-PCR showed that cingulin mutation caused a further increase in the transcript levels of occludin, claudin-2, claudin-6 and claudin-7, which were probably due to an increase in expression of GATA-6, GATA-4 and HNF-4α, transcription factors implicated in endodermal differentiation. Thus, lack of junctional cingulin does not prevent tight-junction formation, but gene expression and tight junction protein levels are altered by the cingulin mutation.
Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle
Authors: Lidonnici Maria Rosa , Rossi Rossella , Paixão Sonia , Mendoza-Maldonado Ramiro , Paolinelli Roberta , Arcangeli Caterina , Giacca Mauro , Biamonti Giuseppe , Montecucco Alessandra
Published: September 2004

In eukaryotes, initiation of DNA replication requires the activity of the origin recognition complex (ORC). The largest subunit of this complex, Orc1p, has a critical role in this activity. Here we have studied the subnuclear distribution of the overexpressed human Orc1p during the cell cycle. Orc1p is progressively degraded during S-phase according to a spatio-temporal program and it never colocalizes with replication factories. Orc1p is resynthesized in G1. In early G1, the protein is distributed throughout the cell nucleus, but successively it preferentially associates with heterochromatin. This association requires a functional ATP binding site and a protein region partially overlapping the bromo-adjacent homology domain at the N-terminus of Orc1p. The same N-terminal region mediates the in vitro interaction with heterochromatin protein 1 (HP1). Fluorescence resonance energy transfer (FRET) experiments demonstrate the interaction of human Orc1p and HP1 in vivo. Our data suggest a role of HP1 in the recruitment but not in the stable association of Orc1p with heterochromatin. Indeed, the subnuclear distribution of Orc1p is not affected by treatments that trigger the dispersal of HP1.
Xin repeats define a novel actin-binding motif
Authors: Pacholsky Dirk , Vakeel Padmanabhan , Himmel Mirko , Löwe Thomas , Stradal Theresia , Rottner Klemens , Fürst Dieter O. , van der Ven Peter F. M.
Published: September 2004

Xin is a protein that is expressed during early developmental stages of cardiac and skeletal muscles. Immunolocalization studies indicated a peripheral localization in embryonic mouse heart, where Xin localizes with β-catenin and N-cadherin. In adult tissues, Xin is found primarily in the intercalated discs of cardiomyocytes and the myotendinous junctions of skeletal muscle cells, both specialized attachment sites of the myofibrillar ends to the sarcolemma. A large part of the Xin protein consists of unique 16 amino acid repeats with unknown function. We have investigated the characteristics of the Xin repeats by transfection experiments and actin-binding assays and ascertained that, upon expression in cultured cells, these repeats bind to and stabilize the actin-based cytoskeleton. In vitro co-sedimentation assays with skeletal muscle actin indicated that they not only directly bind actin filaments, but also have the capability of arranging microfilaments into networks that sediment upon low-speed centrifugation. Very similar repeats were also found in `Xin-repeat protein 2' (XIRP2), a novel protein that seems to be expressed mainly in striated muscles. Human XIRP2 contains 28 Xin repeats with properties identical to those of Xin. We conclude that the Xin repeats define a novel, repetitive actin-binding motif present in at least two different muscle proteins. These Xin-repeat proteins therefore constitute the first two members of a novel family of actin-binding proteins.
The GIN4 family kinase, Cdr2p, acts independently of septins in fission yeast
Authors: Morrell Jennifer L. , Nichols Connie B. , Gould Kathleen L.
Published: September 2004

Two relatives of the GIN4 protein kinase family, Cdr1p and Cdr2p, exist in the yeast Schizosaccharomyces pombe. Although in Saccharomyces cerevisiae GIN4-related kinases influence septin ring organization and septin rings influence the localization and function of GIN4-related protein kinases, it is unknown whether this relationship is conserved in S. pombe. Here, we have probed the relationship between Cdr2p activity and septins and find that Cdr2p and septins are functionally independent. Cdr2p localizes in a cortical band overlying the nucleus during interphase, whose dimension is proportional to cell length, and to a medial ring structure in late mitosis. Both localizations are septin-independent and disrupted by treatment with filipin. Structure/function analysis indicates that the intracellular targeting domain of Cdr2p is largely contained within its non-catalytic C-terminus. Cdr2 protein kinase activity, while unimportant for its localization, is critical for its cell cycle function. Our data indicate that Cdr2p functions at two positions within the cell at discrete cell cycle stages to influence the timing of mitotic entry and cytokinesis, respectively.
Increased importin-β-dependent nuclear import of the actin modulating protein CapG promotes cell invasion
Authors: De Corte Veerle , Van Impe Katrien , Bruyneel Erik , Boucherie Ciska , Mareel Marc , Vandekerckhove Joël , Gettemans Jan
Published: September 2004

CapG (gCap39) is a ubiquitous gelsolin-family actin modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis and motility. CapG is the only gelsolin-related actin binding protein that localizes constitutively to both nucleus and cytoplasm. Structurally related proteins like severin and fragmin are cytoplasmic because they contain a nuclear export sequence that is absent in CapG. Increased CapG expression has been reported in some cancers but a causal role for CapG in tumour development, including invasion and metastasis, has not been explored. We show that moderate expression of green fluorescent protein-tagged CapG (CapG-EGFP) in epithelial cells induces invasion into collagen type I and precultured chick heart fragments. Nuclear export sequence-tagged CapG-EGFP fails to induce invasion, whereas point mutations in the nuclear export sequence permitting nuclear re-entry restore cellular invasion. Nuclear import of CapG is energy-dependent and requires the cytosolic receptor importin β but not importin α. Nuclear CapG does not possess intrinsic transactivation activity but suppresses VP16 transactivation of a luciferase reporter gene in a dose-dependent manner. Furthermore, invasion requires signalling through the Ras-phosphoinositide 3-kinase pathway and Cdc42 or RhoA, but not Rac1. We show for the first time active nuclear import of an actin binding protein, and our findings point to a role for nuclear CapG in eliciting invasion, possibly through interfering with the cellular transcription machinery.
Ultrathin Silica Films with a Nanoporous Monolayer
Authors: Tanaka Shunsuke , Nishiyama Norikazu , Hayashi Yasushi , Egashira Yasuyuki , Ueyama Korekazu
Published: September 2004

Ultrathin silica films with a monolayer of uniform nanopores were fabricated on a silicon substrate by contacting triblock copolymer films with tetraethoxysilane vapor followed by calcination to remove the copolymer.
The Websites of Norwegian hospitals: do they meet national guidelines and patients' expectations?
Authors: Norum Jan , Moen Mari-Ann
Published: September 2004

In 2001, the Norwegian National Health Administration introduced a system that allows patients to choose the public hospital at which they are to be treated. This increased the importance of hospital Websites, as they are used by patients to select a service. In 2002 we rated the Websites of general hospitals in Norway in two ways. Rating 1 was based on a Norwegian set of quality criteria for Websites; 16 items were evaluated to give a maximum score of 32. For rating 2, we used the top five items ranked by cancer patients or their relatives in a previous study, which were weighted and gave a maximum score of 27. Fifty-four (82%) of the 66 public general hospitals in Norway had a Website on the Internet. Information about departments and treatments offered was sparse. Information on prices was almost completely absent. About a third of the Websites were searchable. Quality, as scored by summing ratings 1 and 2, was related to the type of hospital. The mean scores were: university hospitals, 38.8; central hospitals, 30.3; local hospitals, 29.2; and other hospitals, 21.1. On the whole, the hospitals' Websites were not impressive, in view of the competition between hospitals for patients.
In-service nursing education delivered by videoconference
Authors: de Godoy Simone , Costa Mendes Isabel Amélia , Hayashida Miyeko , Nogueira Maria Suely , Marchi Alves Leila Maria
Published: September 2004

Thirty nursing auxiliaries at a Brazilian hospital were trained in intramuscular injection at the ventrogluteal site in a lecture delivered by videoconference (over an IP connection at up to 512 kbit/s). Participants were able thereafter to practise on the same manikins used for demonstration during the lecture, until they felt confident in carrying out the procedure. They then completed an evaluation questionnaire on the use of videoconferencing as a teaching method. Most of their responses were favourable. The principal reasons given for attending the videoconference lecture were the ability to combine work and education, the opportunity to participate in an innovative approach and an interest in the subject of the lecture. The present study demonstrates the potential of videoconferencing for distance education in nursing.
T.120 application sharing for the remote configuration of prostheses
Authors: Lemaire Edward D. , Smith Craig , Nielen David , Fawcett Jocelyn
Published: September 2004

We conducted a study to determine whether application sharing and desktop videoconferencing using the Internet constituted a viable method for remotely configuring a microprocessor-controlled lower-extremity prosthesis. A DSL broadband connection (operated at 256 kbit/s) was used to connect two prosthetic care facilities over the public Internet. PC-based videoconferencing was used with application sharing for the remote configuration of the prosthesis and to view information on the load and angle of the prosthetic knee. Fifteen people with trans-femoral amputations had the settings for their prosthesis scrambled before a specialist at another site remotely configured the device. There were no significant differences between the original and online settings. Clinicians at both sites considered that 14 out of 15 sessions were successful (one trial involved technical problems with the knee unit that were unrelated to the telemedicine trial). The clients rated their satisfaction with the technique as excellent (47%), above average (33%) or average (20%). The present study shows that a combination of desktop videoconferencing and T.120 application sharing can be used for the remote configuration of assistive devices.
Mobile phone text messaging in the management of diabetes
Authors: Ferrer-Roca O. , Cárdenas A. , Diaz-Cardama A. , Pulido P.
Published: September 2004

We conducted a trial of mobile phone text messaging (short message service; SMS) for diabetes management. In an eight-month period, 23 diabetic patients used the service. Patients used SMS to transmit data such as blood glucose levels and body weight to a server. The server automatically answered with an SMS acknowledgement message. A monthly calculated glycosylated haemoglobin result was also automatically sent to the patient by SMS. During the trial the patients sent an average of 33 messages per month. Although users showed good acceptance of the SMS diabetes system, they expressed various concerns, such as the inability to enter data from previous days. Nonetheless, the trial results suggest that SMS may provide a simple, fast and efficient adjunct to the management of diabetes. It was particularly useful for elderly persons and teenagers, age groups that are known to have difficulty in controlling their diabetes.
Stability of Sufentanil Citrate with Levobupivacaine HCl in NaCl 0.9% Infusion After Microwave Freeze–Thaw Treatment
Authors: Boitquin Laurence P , Hecq Jean-Daniel , Vanbeckbergen Danielle F , Jamart Jacques , Galanti Laurence M
Published: September 2004

BACKGROUND

Sufentanil is a powerful central anesthetic and analgesic of the opiate family that can be used with levobupivacaine for epidural administration. Advance preparation of intravenous solution could be useful to improve quality, time management, and cost-savings of drug delivery.

OBJECTIVE

To investigate how freezing, long-term storage, and microwave thawing can affect the stability of sufentanil citrate and levobupivacaine HCl in NaCl 0.9% (saline) (w/v) polyvinyl chloride (PVC) bags.

METHODS

The stability of 5 bags of solution containing sufentanil 500 μg and levobupivacaine 625 mg per 500 mL of saline solution in PVC bags was studied after freezing for 4 months at −30 °C, thawing in a microwave oven with a validated cycle, and storage at 4 °C. The concentrations of the drugs were measured by HPLC using a reversed-phase column, a mobile phase consisting of 18% acetonitrile (v/v) in tertabutyl ammonium hydrogen sulfate buffer 0.03 M pH 3.00 ± 0.05, and UV detection at 235 nm for sufentanil and 260 nm for levobupivacaine. Visual inspection and pH measurement were also performed.

RESULTS

No color change or precipitation was observed. Sufentanil and levobupivacaine were stable for at least 70 days at 4 °C after freezing and thawing. Throughout this period, the 95% lower confidence limit of the concentration–time profile remained >90% of the initial concentration. During this period, the pH value remained stable.

CONCLUSIONS

Within the limits defined here, a mixture of sufentanil citrate and levobupivacaine HCl may be prepared in advance and kept frozen.
Molecular Models of Acidic PePtides From Pea Bud Chromatin And Seminal Plasma. Divalent Cations-Mediated Interaction With Dna
Authors: Mancinelli L. , Chillemi F. , Cardellini E. , Marsili V. , Giavarini F. , Angelis L. DE , Lugaro G. , Gianfranceschi G.L.
Published: September 2004

Small acidic peptides have been isolated from biological fluids (blood and seminal plasma) and from chromatin of several tissues. Their biological activity is related to the control of cell growth and gene expression.

This work is an approach to the study of peptide structure-function relationship. Purified fractions from seminal plasma and pea bud chromatin were subjected to fast ion bombardment mass spectrometry. The results obtained were analyzed according to biochemical characteristics of the peptides studied and some possible molecular models have been designed. Two of the proposed sequences were synthesized and their biological activity assayed in cells and cell-free systems. The results demonstrate that the synthetic peptides are able to bind to DNA in the presence of divalent cations (Mg
Cytidine Triphosphate Synthase Activity and mRNA Expression in Normal Human Blood Cells
Authors: Verschuur A.C. , Gennip A.H. Van , Muller E.J. , Voûte P.A.T. , Vreken P. , Kuilenburg A.B.P. Van
Published: September 2004

Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood cells isolated from healthy volunteers by density centrifugation and elutriation centrifugation. We also investigated the mRNA expression of CTP synthase in lymphocytes and monocytes. The highest activity of CTP synthase was found in thrombocytes (6.48 nmol CTP × mg−1× h−1), followed by that of monocytes (2.23), lymphocytes (1.69), granulocytes (0.52) and erythrocytes (0.42). The activity of CTP synthase in whole blood samples was at an intermediate level (1.27). The mRNA expression of CTP synthase in monocytes was comparable to that observed in lymphocytes.
Activation of Protein C by Arginine-Specific Cysteine Proteinases (Gingipains-R) from Porphyromonas gingivalis
Authors: Hosotaki K. , Imamura T. , Potempa J. , Kitamura N. , Travis J.
Published: September 2004

In order to determine the effect of bacterial proteinases on activation of the protein C system, a negative regulator of blood coagulation, two arginine-specific cysteine proteinases (gingipains R) fromPorphyromonas gingivalis, a causative bacterium of adult periodontitis, were examined. Each enzyme activated human protein C in a dose- and incubation time-dependent manner. Interestingly, the form of enzyme being composed of a non-covalent complex containing both catalytic and adhesion domains (RgpA) produced activated protein C 14-fold more efficiently than RgpB which contained the catalytic domain alone. Thekcat/Kmvalue of RgpA was 18-fold higher than that of RgpB and comparable to that of the thrombin-thrombomodulin complex, the physiological activator of protein C. RgpA catalyzed protein C activation was augmented 1.4-fold by phospholipids, ubiquitous cell membrane components. Furthermore, RgpA, but not RgpB, could activate protein C in plasma and this resulted in a decrease of the protein C concentration in plasma, which is often observed in patients with sepsis during the development of disseminated intravascular coagulation (DIC). These data indicate that RgpA is a more potent activator of protein C than RgpB and suggest that only the former enzyme can cause protein C activationin vivo. The present study further suggests that bacterial proteinases may possibly contribute to the consumption of plasma protein C which predisposes to DIC and/or promotes a thrombotic tendency towards DIC in sepsis.