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PubMed2005

The C2B domain of rabphilin directly interacts with SNAP-25 and regulates the docking step of dense core vesicle exocytosis in PC12 cells.

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) J. Biol. Chem. 279, 13065-13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (i.e. Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca(2+) (EC(50) = 0.817 microm SNAP-25), but 0.5 mm Ca(2+) increases the affinity for SNAP-25 2-fold (EC(50) = 0.405 microm SNAP-25) without changing the B(max) value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-DeltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.

Animals↗
PubMed2006

The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor.

Sperm-specific phospholipase C-zeta (PLCzeta) causes intracellular Ca(2+) oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCzeta, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCzeta mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374-381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg(376), Lys(377), Arg(378), Lys(379), or Lys(381) with glutamate, whereas Ca(2+) oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys(299) and/or Lys(301) in the C terminus of X domain, or Trp(13), Phe(14), or Val(18) in the N terminus of EF1. Ca(2+) oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374-383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296-309 or 1-19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2-4 or C2 domain. Thus, PLCzeta is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCdelta1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.

Active Transport, Cell Nucleus↗
PubMed2000

Agonist-induced isomerization in a glutamate receptor ligand-binding domain. A kinetic and mutagenetic analysis.

Agonist binding to glutamate receptor ion channels occurs within an extracellular domain (S1S2) that retains ligand affinity when expressed separately. S1S2 is homologous to periplasmic binding proteins, and it has been proposed that a Venus flytrap-style cleft closure triggers opening of glutamate receptor ion channels. Here we compare the kinetics of S1S2-agonist binding to those of the periplasmic binding proteins and show that the reaction involves an initial rapid association, followed by slower conformational changes that stabilize the complex: "docking" followed by "locking." The motion detected here reflects the mechanism by which the energy of glutamate binding is converted into protein conformational changes within S1S2 alone. In the intact channel, these load-free conformational changes are harnessed and possibly modified as the agonist binding reaction is used to drive channel opening and subsequent desensitization. Using mutagenesis, key residues in each step were identified, and their roles were interpreted in light of a published S1S2 crystal structure. In contrast to the Venus flytrap proposal, which focuses on motion between the two lobes as the readout for agonist binding, we argue that smaller, localized conformational rearrangements allow agonists to bridge the cleft, consistent with published hydrodynamic measurements.

Amino Acid Substitution↗
PubMed2001

Consumption of bivalve molluscs in Italy: estimated intake of cadmium and lead.

Concentrations of cadmium and lead were determined in nine different species of mollusc bivalves (Modiolus barbatus, Venus verrucosa, Scapharca inaequivalvis, Tapes decussatus, Callista chione, Pecten jacobeus, Ensis siliqua, Venus gallina, Cardium tubercolata) collected from different coastal areas of the Adriatic Sea (north, middle and south Adriatic). The levels of cadmium and lead found in bivalves from the north Adriatic Sea were significantly (P < 0.001) higher than those detected in those from the middle and south Adriatic Sea. In some species of molluscan bivalves (M. barbatus and T. decussatus) from the north Adriatic Sea, concentrations of cadmium and lead exceeded the maximum limit (2 mg/kg w.w.) established by the Italian legislation. Weekly intakes were estimated and compared with the Provisional Tolerable Weekly Intake (PTWI) recommended by the FAO/WHO Expert Committee on Food Additives.

Analysis of Variance↗
PubMed1962

Some pharmacological properties of brain ganglioside.

The effects of a preparation of purified brain ganglioside and of neuraminic acid have been studied on various pharmacological test preparations. In concentrations of 10 mug/ml. or more, ganglioside stimulates the isolated guinea-pig ileum; graded responses are usually obtained. Various substances, known to antagonize the actions of other stimulant substances, fail to affect the response to ganglioside. Ganglioside and neuraminic acid neither stimulate the superior cervical ganglion of the cat nor do they affect ganglionic transmission. Ganglioside and neuraminic acid did not change the response of the frog rectus abdominis muscle to acetylcholine, nor did they affect the blood pressure of the cat. Ganglioside (2 mug/ml. or more) stimulates the isolated heart of Venus mercenaria, but the response, which develops slowly, decreases with repeated administration of ganglioside. The response is not blocked by 2-bromo-lysergic acid diethylamide and there is no indication that it is due to the liberation of endogenous 5-hydroxytryptamine or related indole compounds. For both the heart of Venus mercenaria and guinea-pig ileum, the activity demonstrated by brain ganglioside is specifically a property of the whole molecule, since neither neuraminic acid itself nor a preparation of brain ganglioside from which only two-thirds of its neuraminic acid had been removed are active.

Acetylcholine↗
PubMed2006

Prevention of ulcers by esomeprazole in at-risk patients using non-selective NSAIDs and COX-2 inhibitors.

OBJECTIVES: Proton pump inhibitors reduce ulcer recurrence in non-steroidal anti-inflammatory drug (NSAID) users, but their impact in at-risk ulcer-free patients using the current spectrum of prescribed agents has not been clearly defined. We assessed esomeprazole for ulcer prevention in at-risk patients (> or = 60 yr and/or ulcer history) taking NSAIDs, including COX-2 inhibitors. Such studies are particularly relevant, given that concerns regarding adverse cardiovascular outcomes among COX-2 inhibitor users may prompt re-evaluation of their use. METHODS: We conducted two similar double-blind, placebo-controlled, randomized, multicenter studies; VENUS (United States) and PLUTO (multinational). A total of 844 and 585 patients requiring daily NSAIDs, including COX-2 inhibitors were randomized to receive esomeprazole (20 or 40 mg) or placebo, daily for 6 months. RESULTS: In the VENUS study, the life table estimated proportion of patients who developed ulcers over 6 months (primary variable, intent-to-treat population) was 20.4% on placebo, 5.3% on esomeprazole 20 mg (p < 0.001), and 4.7% on esomeprazole 40 mg (p < 0.0001). In the PLUTO study, the values were 12.3% on placebo, 5.2% with esomeprazole 20 mg (p = 0.018), and 4.4% with esomeprazole 40 mg (p = 0.007). Significant reductions were observed for users of both non-selective NSAIDs and COX-2 inhibitors. Pooled ulcer rates for patients using COX-2 inhibitors (n = 400) were 16.5% on placebo, 0.9% on esomeprazole 20 mg (p < 0.001) and 4.1% on esomeprazole 40 mg (p= 0.002). Esomeprazole was well tolerated and associated with better symptom control than placebo. CONCLUSIONS: For at-risk patients, esomeprazole was effective in preventing ulcers in long-term users of NSAIDs, including COX-2 inhibitors.

Aged↗
PubMed1995

[Bacteremia with Acinetobacter species--clinicopathological characteristics of 27 cases].

Acinetobacter spp. are non-fermented gram-negative rods that are widespread in the environment and colonize in the human skin. They are known to be a nosocomial pathogen causing, pneumonia, meningitis and bacteremia. Recently, they have been found increasingly in catheter-related infections (CRI). Thirty-seven cases of bacteremia were developed in our hospital during the past five years. Of these 27 cases were chosen out of the medical records for discussion in this paper. Twenty-three cases are blood positive for Acinetobacter anitratus and 4 cases for A. lwoffii. Most cases have an underlying disease like hematological malignancy, solid tumor and infantile congenital abnormality. There were also some clinical signs; high fever, hypotension, tachycardia, tachypnea, peripheral cyanosis. A central venus catheter was inserted in 22 cases, and in 13 of these, the catheter was removed after the bacteremic episode. Nine cases became afebrile after the removal of the catheter and A. anitratus was isolated from the catheter tip in four cases. Heparin was administered through the catheter in 7 cases. Formerly Acinetobacter spp. were not recognized as a major pathogen, but recently found increasingly in CRI. We also found 9 cases which were definitely diagnosed or suspected as CRI, and were successfully treated by removal of the central venus catheter. Association between administration of heparin and bacteremia of Acinetobacter spp. was reported, we actually detected such association in 7 cases, but the potential role of heparin has not been clarified yet. Compared with A. lwoffii, A. anitratus were resistant to many drugs, but had good susceptibility to imipenem, minocycline, aminoglycoside, and fluoroquinolone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acinetobacter↗
PubMed2005

High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay.

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2- Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.

Animals↗
PubMed2000

Centers needed to study women's environmental health.

The view of women as primarily fecund beings goes back to prehistory, where it is expressed in the well-known series of Venuses--stone figures of women with enlarged breasts, who are often represented as pregnant. Although the Venus figures date from the late Paleolithic era, this view of women did not change much in the next 20,000 years. With the approaching millennium, however, the field of health research has begun to consider women apart from their children or prospective progeny. Reflecting this shift in viewpoint, funds for research on the environmental health of women have now become available. However, no coordinated program has been launched on the scale of the newly established Centers for Children's Environmental Health and Disease Prevention Research (1). Should women, like children, be the focus of a concerted research effort?

Adult↗
PubMed2004

Generation of optimized yellow and red fluorescent proteins with distinct subcellular localization.

Fluorescent proteins (FPs) have revolutionized many aspects of cell biology and have become indispensable research tools. Today's increasingly complex experiments aiming to understand biological systems strongly depend on the availability of combinations of multiple FPs, which allow their distinguishable simultaneous detection in the same cell or tissue. Recently, the VENUS and DsRed. T4 FPs were described as the latest generation of yellow and red FPs. To increase the combinatorial possibilities when using these optimized FPs, we have generated and successfully tested seven new forms of VENUS and DsRed. T4 proteins with distinct subcellular localization. To facilitate their use as markers in biological experiments, bicistronic expression constructs, which have been optimized for robust expression in almost all mammalian developmental stages and cell types, were produced for the new FPs. In addition, several plasmids were created, which contain all necessary elements for inserting the reading frames of these FPs into specific gene loci in knock-in experiments without disrupting the reading frame of the endogenous gene.

Cell Line↗
PubMed2006

Sustained exposure to high glucose concentrations modifies glucose signaling and the mechanics of secretory vesicle fusion in primary rat pancreatic beta-cells.

The mechanism(s) by which chronic hyperglycemia impairs glucose-stimulated insulin secretion is poorly defined. Here, we compare the "nanomechanics" of single exocytotic events in primary rat pancreatic beta-cells cultured for 48 h at optimal (10 mmol/l) or elevated (30 mmol/l) glucose concentrations. Cargo release was imaged by total internal reflection fluorescence microscopy of lumen-targeted probes (neuropeptide Y [NPY]-pH-insensitive yellow fluorescent protein [NPY-Venus] or NPY-monomeric red fluorescent protein), while the fate of the vesicle membrane was reported simultaneously with phosphatase-on-the-granule-of-insulinoma-enhanced green fluorescent protein. Under all conditions studied, exocytosis proceeded via a "cavity recapture" mechanism in which the vesicle and plasma membranes fused transiently. While essentially complete release of NPY-Venus was observed in 24 +/- 1% of glucose-stimulated exocytotic events in cells maintained at 10 mmol/l glucose, this value was reduced reversibly to 5 +/- 2% of events by culture at 30 mmol/l glucose, in line with decreases in Glut2 and glucokinase gene expression, and attenuated glucose-stimulated increases in NADPH and intracellular [Ca2+]. Since vesicle release in response to cell depolarization with KCl was not affected by culture at 30 mmol/l glucose, we conclude that hyperglycemia causes the abnormal termination of individual insulin release events principally by inhibiting glucose signaling.

Animals↗
PubMed1988

[Participation in a simple large-scale randomized trial organized by EORTC through an international computer network].

Case registration and treatment allocation was achieved in a "Simple large-scale trial against colorectal cancer" designed by the European Organization for Research and Treatment of Cancer (EORTC) using an international computer network system VENUS-P. The access to the central computer "EuroCODE" was obtained by simple equipment including ordinary microcomputer, modem and telephone line. Subscription to the KDD data network VENUS-P using a Packet Assembler/Disassembler (PAD) was necessary to connect to the EuroCODE. Intraoperative patient registration and allocation of treatment were possible through this system regardless of the time difference between countries. To organize a large-scale trial as a means to detect moderate but medically worthwhile treatment differences, this method proved to be particularly effective especially for international cooperative studies.

Colonic Neoplasms↗
PubMed1979

Early Dutch contributions on Peyronie's disease.

Long before De la Peyronie's description of three cases of plastic induration and curvature of the penis (1743) the disorder was already known. The Ephemerides (1687) and the rather scabrous booklet, Venus minzieke gasthuis (Venus Hospital for the lovesick)(1688), contain short remarks on this condition. Two extensive Dutch contributions -- not mentioned in the literature on this subject -- were written by Nicolaas Tulp (1593--1674) and Fredrik Ruysch (1638--1731). The first one is a case history, the second one is more general discussion with a fine engraving added. Treatment varied according to the various early authors. The therapeutic successes claimed must be seen in the light of the natural history of this disease: spontaneous improvement occurs not infrequently. To change the eponym 'Peyronie's disease', however, seems unjustified. It is an homage to François Gigot de la Peyronie (1678-1747) surgeon to King Louis XV and founder of the French Académie de Chirurgie.

France↗
PubMedDate not supplied

[The Temple of Aesculapius in Diocletian's palace in Split].

Many temples, sanctuaries and even the entire small towns have been dedicated to Aesculapius's, the god of medicine. The remnants of Aesculapius temples in the Republic of Croatia have been found on locations of Narona, Salona, Pola and in Diocletian's palace in Split. The first archbishop of Split, "Giovanni di Ravenna", at the end of the 8th century or at the beginning of the 9th century, gave to Diocletian's Mausoleum the name of "Templum Jouis" and reassigned it to Christian church. Thomas the Archdeacon in the 13th century mentioned three temples in Diocletian's Palace: "templa...Jouis, Asclepii, Martis", while in 1567 Antonio Proculiano described four temples: the octogonal temple on the east dedicated to Jupiter (the present cathedral), the rectangular temple on the west (the present baptistry), a minor round temple on the south and a round-hexagonal temple on the north. The majority of authors, for centuries, called the baptistry: Aesculapius's temple, while only a few others called it Jupiter's or Janu's temple. It seems logical to assume that there was only one Jupiter's temple in Diocletian's Palace, namely the present cathedral. Four cultic edifices within the Diocletian's Palace were given six names: "Jouis, Asclepii, Martis, Janus's Kibela's and Venus's. Should the recent research prove the baptistry not to be Aesculapius's temple, the question might be raised whether one of the two, recently discovered small temples, Kibela's or Venus's, was dedicated to Aesculapius?

Croatia↗
PubMed1995

Comets, impacts, and atmospheres.

We are proposing a model for the delivery of volatiles to the inner planets by icy planetesimals (comets). Laboratory studies of the trapping of gases in ice forming at low temperatures simulate the formation of comet nuclei at various distances from the Sun in the solar nebula. The total gas content as well as the relative proportions of gases trapped in the ice are strong functions of temperature. As they trap N2 inefficiently, all planetesimals formed interior to Neptune are deficient in nitrogen, acquiring values of C/N resembling those found in the inner planet volatile inventories. A mixture of three basic types of comets appears capable of accounting for the observed volatile inventories on Venus, Earth, and Mars, with the caveat that impact erosion is necessary to explain the present condition of the martian atmosphere. The model includes the possibility of several epochs of clement conditions on early Mars. Some tests of these ideas are suggested, including measurements in Jupiter's atmosphere by the Galileo probe.

Atmosphere↗
PubMedDate not supplied

Contributions of icy planetesimals to the Earth's early atmosphere.

Laboratory experiments on the trapping of gases by ice forming at low temperatures implicate comets as major carriers of the heavy noble gases to the inner planets. These icy planetesimals may also have brought the nitrogen compounds that ultimately produced atmospheric N2. However, if the sample of three comets analyzed so far is typical, the Earth's oceans cannot have been produced by comets alone, they require an additional source of water with low D/H. The highly fractionated neon in the Earth's atmosphere may also indicate the importance of non-icy carriers of volatiles. The most important additional carrier is probably the rocky material comprising the bulk of the mass of these planets. Venus may require a contribution from icy planetesimals formed at the low temperatures characteristic of the Kuiper Belt.

Earth, Planet↗
PubMed2002

Circular polarization of light by planet Mercury and enantiomorphism of its surface minerals.

Different mechanisms for the generation of circular polarization by the surface of planets and satellites are described. The observed values for Venus, the Moon, Mars, and Jupiter obtained by photo-polarimetric measurements with Earth based telescopes, showed accordance with theory. However, for planet Mercury asymmetric parameters in the circular polarization were measured that do not fit with calculations. For BepiColombo, the ESA cornerstone mission 5 to Mercury, we propose to investigate this phenomenon using a concept which includes two instruments. The first instrument is a high-resolution optical polarimeter, capable to determine and map the circular polarization by remote scanning of Mercury's surface from the Mercury Planetary Orbiter MPO. The second instrument is an in situ sensor for the detection of the enantiomorphism of surface crystals and minerals, proposed to be included in the Mercury Lander MSE.

Astronomy↗
PubMed2001

Assessing the plausibility of life on other worlds.

As the field of astrobiology matures and search strategies for life on other worlds are developed, the need to analyze in a systematic way the plausibility for life on other planetary systems becomes increasingly apparent. We propose the adoption of a simple plausibility of life (POL) rating system based on specific criteria. Category I applies to any body shown to have conditions essentially equivalent to those on Earth. Category II applies to bodies for which there is evidence of liquid water and sources of energy and where organic compounds have been detected or can reasonably be inferred (Mars, Europa). Category III applies to worlds where conditions are physically extreme but possibly capable of supporting exotic forms of life unknown on Earth (Titan, Triton). Category IV applies to bodies that could have seen the origin of life prior to the development of conditions so harsh as to make its perseverance at present unlikely but conceivable in isolated habitats (Venus, Io). Category V would be reserved for sites where conditions are so unfavorable for life by any reasonable definition that its origin or persistence there cannot be rated a realistic probability (the Sun, gas giant planets). The proposed system is intended to be generic. It assumes that life is based on polymeric chemistry occurring in a liquid medium with uptake and degradation of energy from the environment. Without any additional specific assumptions about the nature of life, the POL system is universally applicable.

Biological Evolution↗