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Voltage-dependent N-type Ca2+ channel activity regulates the interaction between FGF-1 and S100A13 for stress-induced non-vesicular release.

1. The Ca(2+)-mediated regulation of interaction between FGF-1 and S100A13 in NG108-15 cells was studied. When the stress by depriving B27 supplement from the culture was given, cellular levels of both proteins were decreased, while their releases were significantly increased within 3 h. These stress-induced changes were all abolished by amlexanox, an anti-allergic drug. 2. These releases were significantly inhibited by the addition of EGTA or BAPTA-AM, cellular or extracellular Ca(2+)-chelating agent, respectively. The addition of omega-conotoxin GVIA, a N-type Ca(2+)-channel blocker caused a complete inhibition of the release, while increased the cytosolic levels of both proteins. However, omega-conotoxin MVIIC, the non-N-type Ca(2+)-channel blocker was ineffective. 3. In NG108-15 cells, which had been transfected with Venus-FGF-1 and CFP-S100A13, the supplement-deprivation stress caused several spike-type fluorescence resonance energy transfer (FRET) signals, suggesting that both proteins showing interaction would be immediately released. These spikes were completely abolished by the addition of omega-conotoxin GVIA. However, the addition of amlexanox caused bell-shaped FRET signals without spikes. 4. Thus, it is suggested that the interaction between FGF-1 and S100A13 responsible for stress-induced non-vesicular release is dependent of Ca(2+)-influx through N-type Ca(2+)-channels.

Aminopyridines↗

Molecular basis of protective effect by crocetin on survival and liver tissue damage following hemorrhagic shock.

Hemorrhagic shock (HS) causes reduction of cellular energy stores, as measured by levels of ATP and ADP. Furthermore, energy depletion may cause mitochondrial damage, which in turn leads to cell death by apoptosis. The hypothesis of the present study is that by enhancing the recovery of cellular ATP and ADP and mitochondrial damage can be reduced, and the extent of apoptosis minimized. Crocetin, a carotenoid compound, appears to enhance the diffusion of oxygen in aqueous solution, and hence may improve energy stores both to the cell and within it. HS was produced in Sprague-Dawley rats by withdrawing blood from the carotid cannula until a mean arterial pressure of 35-40 mm Hg was reached, and then maintained by further withdrawals of blood for 30 and 60 min. Crocetin was administered 2-4 mg/kg in resuscitation fluid through venus cannula and the animals survived for 24-48 h after HS. Experiments designed to promote tissue reconstitution of ATP using crocetin indicate that these approaches are successful in increasing ATP post-hemorrhage and survival. Crocetin treatment also inhibited cellular damage as indicated by increase of Bcl-2 following decrease in cytosolic cytochrome c and caspase-3 after resuscitation. The prolonged energy deficit seen after hemorrhagic shock can produce late damage and rapid restoration of ATP levels to baseline can reduce apoptosis. In conclusions, crocetin can minimize the cellular damage as evidenced by apoptosis and increased the survival of rats.

Adenine Nucleotides↗

Galileo Avionica's technologies and instruments for planetary exploration.

Several missions for planetary exploration, including comets and asteroids, are ongoing or planned by the European Space Agencies: Rosetta, Venus Express, Bepi Colombo, Dawn, Aurora and all Mars Programme (in its past and next missions) are good examples. The satisfaction of the scientific request for the mentioned programmes calls for the development of new instruments and facilities devoted to investigate the body (planet, asteroid or comet) both remotely and by in situ measurements. The paper is an overview of some instruments for remote sensing and in situ planetary exploration already developed or under study by Galileo Avionica Space & Electro-Optics B.U. (in the following shortened as Galileo Avionica) for both the Italian Space Agency (ASI) and for the European Space Agency (ESA). Main technologies and specifications are outlined; for more detailed information please refer to Galileo Avionica's web-site at: http://www.galileoavionica.com .

Astronomy↗

Quality of life of people with venous leg ulcers: a comparison of the discriminative and responsive characteristics of two generic and a disease specific instruments.

BACKGROUND: Venous leg ulcers are an important source of morbidity in society. Measuring the impact of leg ulcers on quality of life is important within clinical and economic evaluations. In this study we report a validation study of the leg ulcer disease specific Hyland questionnaire and compare its discriminative and responsive characteristics to general health quality of life measures: the SF-12 and EQ-5D. METHODS: HRQoL of venous leg ulcer patients from 9 UK regions was measured using SF-12, EQ-5D and Hyland, at baseline and every three months for 1 year. Psychometric analysis was used to confirm the validity of the Hyland questionnaire. Quarterly scores for all instruments were calculated. Effect size and standardised mean difference were used to investigate the responsiveness to ulcer healing and discriminative abilities of the instruments. RESULTS: Three hundred and eighty seven individuals were recruited into the VenUS I study. Baseline health related quality of life data from the study participants suggested a two factor solution for the Hyland. This questionnaire was associated with small and moderate ability to discriminate individuals according to age, mobility, initial ulcer size and ulcer duration. SF-12 and EQ-5D had good evaluative properties; both instruments were responsive to changes in HRQoL after ulcer healing. High levels of bodily pain were reported in the SF-12 questionnaire, whilst only minor ulcer related discomfort was reported in the Hyland. DISCUSSION: SF-12 and EQ-5D are suitable for exploring dimensions of health related quality of life in people with chronic venous leg ulceration. The responsiveness to healing of the Hyland questionnaire is unclear. We would recommend the use of generic instruments for the measurement of HRQoL in patients with venous leg ulcers.

Adult↗

Enzymatic conversion of the antibiotic metronidazole to an analog of thiamine.

We propose that adverse effects of the antibiotic metronidazole may be due, wholly or in part, to its conversion to a thiamine analog and consequent vitamin B1 antagonism. Consistent with this hypothesis, the drug is accepted as a substrate for the thiaminase (EC 2.5.1.2) elaborated as an exoenzyme by the human gut flora constituent Bacillus thiaminolyticus and is also a substrate for the intracellular thiaminase of the mollusk Venus mercenaria. The product, identified as the 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-hydroxyethyl)-2-methyl-4 - nitroimidazolium cation, is a close structural analog of thiamine and is an effective inhibitor of thiamine pyrophosphokinase in vitro. Due to its susceptibility to nucleophilic attack, the analog is unstable, releasing inorganic nitrite under mild conditions. Enzymatic alkylation reactions such as that effected by thiaminase may have general pharmacological significance as a route of increasing the electrophilicity and/or reduction potential of drugs which are heterocyclic weak bases.

Animals↗

Surface-replica topography of retinal pigment epithelium during phagocytosis.

The initial step in retinal phagocytosis may be mediated by complementary recognition molecules on the pigment epithelial microvilli and outer segment membrane surfaces. These molecules may display a surface morphology which is related topographically to phagocytic events. In order to explore this, we have developed a method for replicating the membrane surfaces of rat pigment epithelial explants during phagocytosis of latex beads. The explants are fixed in a mixed aldehyde and osmium fixative and the basal surface glued to a coverslip. After dehydration and critical point drying, the tissue is replicated with platinum and carbon in a freeze-fracture apparatus using either rotary or unidirectional coating and the replicas are examined by transmission electron microscopy. The membrane surfaces of the pigment epithelial cells and their microvilli are studded with numerous surface particles varying in size from 20 to 50 nm that are closely packed and give a cobblestone appearance to the membrane topography. During the early stages of phagocytosis, microvillous processes appear to spread over the beads and form branched processes as they contact bead surfaces. During bead engulfment the microvilli have shortened into flattened sheets with interdigitating processes overlapping the beads. These uptake sites resemble Venus' fly-traps, which as they close over the beads create craters in the membrane surfaces of the pigment epithelium. The crater-like uptake sites appear to flatten out in the later stages and form flattened membrane domains which are surrounded by crenulated membrane that displays an irregular particulate morphology.

Animals↗

Blood flow in the uterine loop artery and loop vein is bidirectional in the mouse: implications for transport of steroids between fetuses.

In rodents, steroids are able to pass between fetuses. Although not previously examined in mice, we have provided evidence that blood flow in the uterine loop artery in rats is bidirectional and that steroids are transported between fetuses by diffusion via the uterine lumen, not via the uterine blood vessels of the mother. The direction of blood flow in the uterine loop artery and vein feeding each uterine horn in house mice was examined on day 17 of pregnancy. Dye was injected into the heart to determine the direction of blood flow in the uterine artery while injection of dye into individual placentae was used to determine the direction of blood flow in the uterine vein. Blood entered the loop artery from both the dorsal and caudal ends and was thus bidirectional. Venus blood flow from placentae was in a rostral direction from placentae in the rostral portion of a uterine horn and in a caudal direction from placentae in the caudal portion of a uterine horn. Comparison of anogenital distance and body weight at birth using a variety of classification schemes, based on different assumptions about the mechanism and direction of transport of steroids between fetuses, showed that the only scheme which accounted for variation in anogenital distance at birth in female mice was that which was based on the number of directly adjacent male siblings in utero. Taken together with recent findings in rats reported elsewhere, we conclude that blood flow in the uterine blood vessels is bidirectional, and steroids are transported between fetuses by diffusing through the amniotic fluid and across the fetal membranes of adjacent fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Asymmetric secretion of principal ovarian venous steroids in the primate luteal phase.

We have characterized the degree of asymmetry of ovarian steroid secretion in the luteal phase of the menstrual cycle in rhesus and cynomolus monkeys. Femoral blood levels of FSH, LH, progesterone, estradiol and 17-hydroxyprogesterone were determined. In addition, laparotomies were performed in the early, mid or late luteal phase to facilitate localization of the corpus luteum and collection of ovarian venus blood. We conclude that: 1) the ovary bearing the active corpus luteum contributes virtually all of the progesterone entering peripheral circulation in the luteal phase; 2) the ipsilateral ovary secretes more 17-hydroxyprogesterone one than the contralateral one, although both are active in the luteal phase; and 3) the asymmetrical secretion of estradiol was manifest only in the early and mid-luteal phase, with ovarian symmetry being reestablished in the late luteal phase.

17-alpha-Hydroxyprogesterone↗

The CO2 greenhouse effect and the thermal history of the atmosphere.

The influence of the expected rise of CO2 content in our atmosphere upon terrestrial temperature is uncertain. A significant increase in temperature could be threatening to certain aspects of terrestrial biology. On the other hand, it is a general consensus among paleobiologists that the Earth possessed a CO2 atmosphere in the past billion years, without dramatic temperature variations endangering the continuity of life. In order to clarify this problem, and to contribute to the understanding of the CO2 greenhouse effect on Venus we have computed the absorption spectrum of CO2 for a wide range of atmospheric concentrations. More than 2500 spectral lines of the 15 micron band were taken into account in our line-by-line calculation. We have used an empirical exponential line-shape function at the line edges. Our results agree with the experimental data of F. W. Taylor. The estimated increase in surface temperature does not reach the boiling point of water even for CO2 concentrations thousands of times larger than the present concentrations. Higher energy (>666 cm-1) CO2 bands and/or an increase in atmospheric H2O may, however, amplify the greenhouse effect.

Atmosphere↗

Indications and counterindications for applying different versions of closed ecosystems for space and terrestrial problems of life support.

Different versions of manned closed ecosystems (CES) based on photosynthesis of unicellular and/or higher plants and chemosynthesis or bacteria are considered. Different versions of CES have been compared for applying them on Earth, Moon, Mars and Venus orbital stations, for Mars missions and planetary stations as well as to provide high-quality life in extreme conditions on the Earth. In microgravity [correction of mycrogravity] we recommend CES with unicellular organisms based on photosynthesis or chemosynthesis (depending of the availability of the light or electric energy). For the planetary stations with Moon gravity and higher CES with higher plants are recommended. Improvement of indoor air quality by CES biotechnology is considered.

Bacteria↗

A high-throughput method for development of FRET-based indicators for proteolysis.

SCAT3 is a fluorescence resonance energy transfer (FRET)-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3-sensitive linker, and an enhanced yellow fluorescent protein with efficient maturation property (Venus). Despite its considerable promise, however, greater responsivity of fluorescence to the proteolysis has been desired for better understanding of spatio-temporal pattern of the activation of caspase-3 during apoptosis. In the present study, the length of linker regions of SCAT3 has been thoroughly optimized by use of a PCR technique. The bacterial colonies expressing the constructs were screened for high FRET efficiency using our home-made fluorescence image analyzer. The FRET signal of an improved SCAT3 changed by about tenfold during apoptotic events in mammalian cells, enabling visualization of caspase-3 activation with better spatial resolution than before. This new high-throughput method will be applicable to development and improvement of FRET-based indicators for proteolysis.

Apoptosis↗

In vivo interaction between RGS4 and calmodulin visualized with FRET techniques: possible involvement of lipid raft.

Regulators of G-protein signaling (RGS) are a family of proteins which accelerate intrinsic GTP-hydrolysis on heterotrimeric G-protein-alpha-subunits. Although it has been suggested that the function of RGS4 is reciprocally regulated by competitive binding of the membrane phospholipid, phosphatidylinositol-3,4,5,-trisphosphate(PtdIns(3,4,5)P(3)), and Ca(2+)/calmodulin (CaM), it remains to be shown that these interactions occur in vivo. Here, using fluorescence resonance energy transfer (FRET) techniques, we show that an elevation of intracellular Ca(2+) concentration by ionomycin increased the FRET efficiency from ECFP (a variant of cyan fluorescent protein)-labeled calmodulin to Venus (a variant of yellow fluorescent protein)-labeled RGS4. The increase in FRET efficiency was greatly attenuated by pre-treating the cells with methyl-beta-cyclodextrin, which depletes membrane cholesterol and thus disrupts lipid rafts. These results provide the first demonstration of a Ca(2+)-dependent interaction between RGS4 and CaM in vivo and show that association in lipid rafts of the plasma membrane might be involved in this physiological regulation of RGS proteins.

Binding Sites↗

An efficient transgenic system by TA cloning vectors and RNAi for C. elegans.

In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusion gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.

Animals↗

Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs.

The binding interface of calmodulin and a calmodulin binding peptide were reengineered by computationally designing complementary bumps and holes. This redesign led to the development of sensitive and specific pairs of mutant proteins used to sense Ca(2+) in a second generation of genetically encoded Ca(2+) indicators (cameleons). These cameleons are no longer perturbed by large excesses of native calmodulin, and they display Ca(2+) sensitivities tuned over a 100-fold range (0.6-160 microM). Incorporation of circularly permuted Venus in place of Citrine results in a 3- to 5-fold increase in the dynamic range. These redesigned cameleons show significant improvements over previous versions in the ability to monitor Ca(2+) in the cytoplasm as well as distinct subcellular localizations, such as the plasma membrane of neurons and the mitochondria.

Amino Acid Sequence↗

Characterization of nanofilled compared to universal and microfilled composites.

OBJECTIVES: The purpose of this study was to compare the inorganic fraction and the mechanical properties of three nanofilled composites with four universal hybrid and two microfilled composites. The degrees of conversion of the materials photopolymerized using halogen and LED units were also investigated. METHODS: Three nanofilled (Supreme, Grandio and Grandio Flow), four universal hybrid (Point-4, Tetric Ceram, Venus, Z 100) and two microfilled (A 110, Durafill VS) composites were used in this study. Their filler weight content was measured by thermogravimetric analysis. The morphology of the filler particles was determined using scanning-electron microscopy (SEM). Mechanical properties were measured: dynamic and static elastic moduli, flexural strength and Vickers microhardness. The degree of conversion in relation with the depth of polymerization of every material tested was evaluated using Raman spectrophotometry. RESULTS: Nanofilled resin composites show higher elastic moduli than those of universal and microfilled composites, except for the Z-100. The microfilled composites exhibit by far the lowest mechanical properties. The flexural strength does not appear as a discriminating factor in this study. The degrees of polymerization obtained with the halogen lamp are higher than those obtained with the LED lamp. SIGNIFICANCE: Nanofilled resin composites show mechanical properties at least as good as those of universal hybrids and could thus be used for the same clinical indications as well as for anterior restorations due to their high aesthetic properties.

Composite Resins↗

A novel analytical method for in vivo phosphate tracking.

Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (P(i)) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows P(i)-dependent increases in FRET efficiency. FLIPPi affinity mutants cover P(i) changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na(+)/P(i) co-transporter exhibited FRET changes when perfused with 100 microM P(i), demonstrating concentrative P(i) uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of P(i) metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P(i) during cell migration.

Animals↗

Freeze-dried formulations for in vivo gene delivery of PEGylated polyplex micelles with disulfide crosslinked cores to the liver.

A stable, freeze-dried formulation consisting of a core-shell-type polyplex with a poly(ethylene glycol) (PEG) shell (polyplex micelles) was prepared from a polyion complex of plasmid DNA (pDNA) and thiolated PEG-poly(L-lysine) block copolymers. The use of lyoprotectants was avoided by crosslinking the core with disulfide bonds. The crosslinked polyplex micelles (CPMs) showed excellent stability during freeze-drying and reconstitution processes, which is in sharp contrast with the formation of visible agglomerates from the non-crosslinked polyplex micelles (NCPMs) after a similar process. A thiolation degree higher than 13% of the lysine residues was required to achieve sufficient tolerability of the CPMs during the freeze-drying/reconstitution cycle. Dynamic light scattering measurements and atomic force microscopy observations demonstrated that the original size and shape of the CPMs with a thiolation degree of higher than 13% were maintained even after the freeze-drying. Furthermore, the CPMs reconstituted from the freeze-dried state achieved a transfection efficiency as high as that of the original samples. The intravenous injection of the CPM with a thiolation degree of 37% into mice via the orbital vein led to an appreciably uniform gene expression of a yellow fluorescence protein variant (Venus) in the liver, while no gene expression was observed in the case of the free pDNA injection. The procedure of disulfide crosslinking of the polyplex micell core allows the preparation of non-viral gene vectors as a powder formulation without the use of any lyoprotectants. This achievement is certainly useful for pharmaceutical applications and exhibits many advantages, including easy concentration adjustments of dosing samples, long-term storage stability, and large-scale production reproducibility.

Animals↗

Crystal structure of Bordetella pertussis BugD solute receptor unveils the basis of ligand binding in a new family of periplasmic binding proteins.

Periplasmic binding proteins of a new family particularly well represented in Bordetella pertussis have been called Bug receptors. One B.pertussis Bug protein is part of a tripartite tricarboxylate transporter while the functions of the other 77 are unknown. We report the first structure of a Bug receptor, BugD. It adopts the characteristic Venus flytrap motif observed in other periplasmic binding proteins, with two globular domains bisected by a deep cleft. BugD displays a closed conformation resulting from the fortuitous capture of a ligand, identified from the electron density as an aspartate. The structure reveals a distinctive alpha carboxylate-binding motif, involving two water molecules that bridge the carboxylate oxygen atoms to the protein. Both water molecules are hydrogen bonded to a common carbonyl group from Ala14, and each forms a hydrogen bond with one carboxylate oxygen atom of the ligand. Additional hydrogen bonds are found between the ligand alpha carboxylate oxygen atoms and protein backbone amide groups and with a threonine hydroxyl group. This specific ligand-binding motif is highly conserved in Bug proteins, indicating that they may all be receptors of amino acids or other carboxylated solutes, with a similar binding mode. The present structure thus unveils the bases of ligand binding in this large family of periplasmic binding proteins, several hundred members of which have been identified in various bacterial species.

Amino Acid Sequence↗