Efficacy and safety of bovine activated factors IIa/VIIa/IXa/Xa in patients with active gastrointestinal bleeding: a proof of concept study.

BACKGROUND AND STUDY AIMS: Endoscopic treatment of active gastrointestinal bleeding often remains difficult, and considerable technical expertise is required. Our aim was to assess the efficacy and safety of endoscopic hemostasis with a liquid combination of bovine activated factors IIa/VIIa/IXa/Xa (SeraSeal). METHODS: Patients with active gastrointestinal bleeding were prospectively included. In group A, 5 mL of bovine activated factors IIa/VIIa/IXa/Xa was topically applied via catheters to the bleeding site as initial hemostasis; group B received a similar application but as rescue therapy after failure of conventional endoscopic hemostasis. RESULTS: In group A, bleeding was stopped by the agent in 15 /22 patients (68 %) and by conventional endoscopic hemostasis in 5 of the other 7, with coiling and surgery required for definitive hemostasis in 2. In group B, the addition of the agent definitively stopped bleeding in 13 /15 patients (87 %), with hemostasis in the remaining 2 achieved with fibrin glue. Rebleeding was observed in 1 patient. CONCLUSIONS: Our proof of concept study suggests that the use of bovine activated factors IIa/VIIa/IXa/Xa might be a safe and effective addition to current endoscopic hemostatic strategies, but further studies are necessary.ClinicalTrials.gov identifier: NCT02349490.

Nanofiber-nanorod composites exhibiting light-induced reversible lower critical solution temperature transitions.

Stimuli-responsive materials are promising as smart materials for a range of applications. In this work, a photo-crosslinkable, thermoresponsive macromer was electrospun into fibrous scaffolds containing gold nanorods (AuNRs). The resulting fibrous nanocomposites composed of poly(N-isopropylacrylamide-co-polyethylene glycol acrylate) (PNPA) and PEGylated AuNRs were crosslinked and swollen in water. AuNRs strongly absorb in the near-infrared (NIR) region to generate heat, which triggered the fiber thermal transition upon NIR light exposure. During the thermal transition, scaffolds collapsed both macroscopically and microscopically, with individual fibers deswelling and pulling together. Exposure to a 1.1 W NIR laser decreased the diameter of swollen fibers by 34.7% from 1332 ± 193.3 to 868.9 ± 168.3 nm, and increased fiber density 116% from 209.5 ± 26.34 to 451.9 ± 23.68 fibers mm( - 1). This transition was dependent on the incorporation of the AuNRs, and was utilized to trigger the release of encapsulated proteins from the nanocomposite fiber mats. The expulsion of water from fibers upon NIR exposure caused the release rate of incorporated protein to increase greater than tenfold, from 0.038 ± 0.052 without external stimulus to 0.462 ± 0.227 µg protein/mg polymer/min with NIR exposure. These results suggest that light-responsive fibrous nanocomposites can be utilized in applications such as drug delivery.

Extracellular calmodulin stimulates light-independent RbcS-GUS expression in suspension-cultured cells of transgenic tobacco.

The RbcS genes encode the small subunits of rubisco; the expression of these genes is controlled in a light-dependent and independent manner. It has been reported that intracellular calmodulin (CaM) is involved in light-dependent RbcS expression. In this report, the role of extracellular CaM in regulating expression of RbcS in darkness was examined. The time course of expression of RbcS-GUS and that of the secretion of CaM in the suspended transgenic tobacco cells in darkness were very similar. Both showed initial increase followed by decline with maximum CaM secretion preceding maximum GUS expression by 24 h. The concentration of CaM in the culture medium is regulated light independently. Purified CaM alone added to the media enhanced RbcS-GUS expression in darkness. The addition of membrane-impermeable CaM inhibitors, such as anti-CaM antiserum or W7-agarose, repressed the expression of RbcS-GUS in darkness, but this inhibitory effect was completely reversed by adding exogenous purified CaM. These results provide the first evidence that extracellular CaM is involved in the regulation of light-independent RbcS gene expression.

Tritiated thymidine and bromodeoxyuridine double-labelling studies on growth factors and oral epithelial proliferation in the mouse.

Mouse tongue epithelium is characterized by a circadian variation in the number of cells undergoing DNA synthesis. Groups of male BDF1 mice were followed over 48 h and a double-labelling method with tritiated thymidine and bromodeoxyuridine used to determine S-phase labelling indices, together with cell influx to and cell efflux from S, at 4-hourly time points. Control animals exhibited diurnal peaks in labelling index at 03:00 with trough activity 12 h later at 15:00. Cell influx peaked at 23:00 with troughs occurring between 11:00 to 15:00. Peak cell efflux occurred at 07:00 with trough activity at 19:00. Animals injected with epidermal growth factor at 05:00 demonstrated a significant fall in both influx and efflux throughout the 48-h period (P < 0.001), but with preservation of labelling indices, suggesting a slower transit of cells through S-phase, whereas epidermal growth factor injected at 15:00 only produced a significant rise in cell-efflux values. Adrenergic stimulation by intravenous phenylephrine/isoprenaline injection at both 05:00 and 15:00 resulted in a significant rise in cell efflux (P < 0.001), although there was also a rise in labelling index in the 15:00 group (P < 0.001). Animals injected with calmodulin at 05:00 demonstrated a significant reduction in labelling index throughout the 48-h period (P < 0.001), but maintained control values for cell influx and efflux, suggesting faster transit of cells through S. Calmodulin injection at 15:00 produced only a significant reduction in cell influx (P < 0.001). Administration of exogenous growth factors significantly alters the normal rhythmical proliferation of oral epithelial cells in a mouse model. These effects appear to be both growth factor- and time-dependent, and may have both physiological and pathological implications.

The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.

Conformation and vasoreactivity of VIP in phospholipids: effects of calmodulin.

The purpose of this study was to determine the conformation and vasorelaxant effects of vasoactive intestinal peptide (VIP) self-associated with sterically stabilized phospholipid micelles (SSM) and whether calmodulin modulates both of these processes. Circular dichroism spectroscopy revealed that VIP is unordered in aqueous solution at room temperature but assumes appreciable a helix conformation in SSM. This conformational transition was amplified at 37 degrees C and by a low concentration of calmodulin (0.1 nM). Suffusion of VIP in SSM elicited significant time- and concentration-dependent potentiation of vasodilation relative to that elicited by aqueous VIP in the in situ hamster cheek pouch (P < 0.05). This response was significantly potentiated by calmodulin (0.1 nM). Collectively, these data indicate that exogenous calmodulin interacts with VIP in SSM to elicit conformational transition of VIP molecule from a predominantly random coil in aqueous environment to alpha helix in SSM. This process is associated with potentiation and prolongation of VIP-induced vasodilation in the in situ peripheral microcirculation.

Calcium-calmodulin-stimulated phosphorylation of rat parotid secretion granule proteins.

In studies designed to determine the mechanism by which Ca++ and calmodulin stimulate the fusion of parotid secretion granules with plasma membrane vesicles, the hypothesis tested was that Ca++ and calmodulin act by stimulating protein phosphorylation. It was earlier found that Ca++ and calmodulin, but neither alone, stimulated the phosphorylation of four secretion granule proteins with molecular masses of 64, 58, 55 and 31 kDa, and decreased the degree of phosphorylation of a 36-kDa protein. Further studies have shown that in the presence of an optimal concentration of calmodulin (2.4 microM), half-maximal activation of phosphorylation of the four proteins occurred at approx. 8 microM Ca++, and at a maximally effective Ca++ concentration (10(-4) M), half-maximal stimulation occurred at calmodulin concentrations between 0.13 and 1.1 microM for the different proteins. The studies now described also demonstrate that the need for calmodulin for stimulating the phosphorylation, but not the dephosphorylation, is specific; two other Ca(++)-binding proteins, parvalbumin and troponin, could not replace calmodulin in stimulating phosphorylation of the four secretion granule proteins, but either one could substitute for calmodulin in stimulating dephosphorylation of the 36-kDa protein. Additionally, the phosphorylated proteins appear to be located on the granule surface. When secretion granules were subjected to mild treatment with a concentration of trypsin that did not lyse the granules, the 31-, 36-, 55-, 58- and 64-kDa proteins were no longer observed. In the presence of optimal concentrations of Ca++ and calmodulin, a dose-dependent inhibition of the phosphorylation of the various proteins by two calmodulin antagonists, trifluoperazine and calmidazolium, was observed; 50% inhibition of phosphorylation of the different proteins was obtained at approx. 20-40 microM trifluoperazine and at about 2.5-3.0 microM calmidazolium. Inhibition of the dephosphorylation of the 36-kDa protein required greater concentrations of trifluoperazine and calmidazolium; 128 microM and 50 microM, respectively. These results are consistent with the hypothesis that the phosphorylation of one or more of the 31-, 55-, 58- and 64-kDa proteins, but not the dephosphorylation of the 36-kDa protein, may be involved in the action of Ca++ and calmodulin in secretion granule-plasma membrane fusion.

Interferon-gamma-induced HLA-DR, but not ICAM-1, expression of human keratinocytes is down-regulated by calmodulin antagonist.

Interferon-gamma (IFN-gamma) has been shown to induce or enhance the expression of MHC class II and intercellular adhesion molecule-1 (ICAM-1) in a variety of human and murine cell types, including epidermal keratinocytes (KC). However, the expression of MHC class II and ICAM-1 molecules induced by IFN-gamma is not necessarily coordinated. We investigated the inhibitory effects of the calmodulin antagonist, W-7, and its chlorine deficient inactive analogue, W-5, on the expression of MHC class II (HLA-DR) and ICAM-1 by human KC incubated with IFN-gamma. We found that the IFN-gamma-induced expression of HLA-DR was reproducibly and dose-dependently inhibited by W-7. However, the expression of ICAM-1 was highly resistant to the inhibitory effects of W-7. Neither HLA-DR nor ICAM-1 expression was affected by W-5. These data suggest that the IFN-gamma-induced HLA-DR, but not ICAM-1, expression is mediated, if not exclusively, by calmodulin in human KC.

Homeostasis intracelular del calcio: la calmodulina y la Ca2+ -ATPasa de la membrana plasmática de tripanosomatidios / Intracellular calcuim homeostasis calmodulin and the plasma menihane Ca2+ ATPase of trypanosomatids

La concentración citoplasmática de calcio iónico en todas las células eucarióticas estudiadas hasta el presente es de 4 órdenes de magnitud menor que la del exterior celular. En tripanosomatidios se ha determinado que la concentración intracelular de este cation es de alrededor de 50 mm, aun menor que la reportada en eucariotes superiores. Esta diferencia de concentración es mantenida mediante diversos sistemas de transporte ubicados a nivel de la membrana plasmática y en ciertos organelos intracelulares. En el caso de los tripanosomatidios, se ha identificado la presencia de un uniporte electroforético en la membrana interna de la mitocondria única gigante de estos parásitos, la cual presenta propiedades cinéticas esencialmente idénticas a las reportadas en aucariotes superiores. Este sistema presenta una afinidad por el Ca2+ incompatible con la posibilidad de mantener la concentración de este cation a nivel submicromolar. Por otra parte, contrario a lo reportado por otros autores, hemos identificado la presencia de una Ca2+ -ATPasa en la membrana plásmatica de Leishmania braziliensis, Leishmania mexicana, Trypanosoma cruzi y Tripanosona brucei. La enzima presenta alta afinidad por Ca2+ (Kmap Ca2+ = 0.5µM), es dependiente de Mg2+, y es estimulante por la calmodulina purificada de los mismos hemoflagelados. Esta Ca2+ -ATPasa es sensible al vanadato (Ki=1µM), lo cual permite identificarla como bomba iónica del tipo "P". Vesículas provenientes la membrana plasmática de estos parásitos son capaces de acumular Ca2+ en contra de un gradiente de concentración. Las características cinéticas de este transporte son similares a las de la Ca2+ -ATPasa, lo cual apoya que se trata de la misma entidad molecular. Los resultados obtenidos permiten postular que la Ca2+ -ATPasa es la estructura responsable del mantenimiento de la concentración intracelular de Ca2+ a nivel submicromolar a largo en estos parásitos

Posibles nuevos moduladores de la bomba de calcio de los eritrocitos humanos / Possible new modulators the Ca pump from human erythrocytes

Se presenta una revisión sobre la Bomba de Ca de los eritrocitos humanos, donde se destacan aspectos generales del ciclo catalítico y el efecto de moduladores naturales, tales como calmodulina, fosfolípidos acídicos y ácidos grasos poli-insaturados, proteólisis por calpaína controlada y proteínquinasas. Se discute el efecto modulador del ATP, iones mono y divalentes, haciendo énfasis en experimentos del autor y colaboradores que señalan al Ca y Mg como posibles nuevos moduladores de la CaATPasa

Calcineurin trapped in liposomes inhibits the action potentials of isolated 3-days-old embryonic chick ventricle.

Calcineurin, an intracellular protein phosphatase (type 2B), is reported to inhibit L-type (slow) calcium channels and thereby play a key role in channel inactivation. The present study was undertaken to examine effects of calcineurin on slow channel dependent action potentials of 3-days-old embryonic chick ventricle and to assess the role of this enzyme in regulation of developing slow channels. Calcineurin trapped in phosphatidylcholine-liposomes to facilitate its intracellular uptake was found to inhibit maximal upstroke velocity (+Vmax), overshoot and duration of action potentials. At higher doses of calcineurin containing liposomes the preparations ceased to exhibit spontaneous activity but elicited electrically driven action potentials with lower +Vmax and overshoot. These observations show that calcineurin down-modulates the embryonic cardiac slow channels under basal conditions.

Ca(2+)-responsive extensible monolayer membrane of calmodulin-albumin conjugate.

A Ca(2+)-responsive monolayer protein membrane was prepared by developing calmodulin and bovine serum albumin at the air-water interface and by conjugating them with a bifunctional agent. In the case of the BSA monolayer, complex formation with Mg2+ generated a larger change in surface pressure than that with Ca2+. On the other hand, a drastic change in surface pressure was observed for the conjugated thin membrane associated with Ca2+ than that associated with Mg2+. Due to a drastic change in the conformation of calmodulin, the conjugated protein film changes its morphology (STM image), depending on Ca2+ concentration: the extended structure in the presence of Ca2+ transforms to a shrinked structure in the absence of Ca2+. The largest surface pressure change was detected when calmodulin was mixed with an equimolar amount of bovine serum albumin.

Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.

The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence. Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically. A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium. Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity. However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule. Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.

The effect of pre- or post-treatment with a calmodulin inhibitor (trifluoperazine) on the response to cytotoxic agents of cells within small EMT6/Ca/VJAC spheroids.

Treatment of small EMT6 spheroids (approximately 250 microns in diameter) with trifluoperazine (TFP), a calmodulin inhibitor, before drug exposure did not alter cellular response to adriamycin (ADM) (5 micrograms/ml), CCNU (5 micrograms/ml) or vincristine (VCR) (1 micrograms/ml). The cell killing effect of nitrogen mustard (HN2) was, however, suppressed by TFP pre-treatment even when the TFP was removed before HN2 exposure. Treatment of small spheroids with TFP for 24 hr after drug exposure was found to have no effect on recovery from potentially lethal damage (PLDR) following bleomycin (BLM) (40 micrograms/ml), CCNU (5 micrograms/ml), HN2 (1 micrograms/ml), or X rays (9 Gy). The surviving fraction measured immediately after drug exposure (SF-0) and the surviving fraction with 24 hr delayed assay (SF-24) for cells within small spheroids were similar following 1 hr exposure to ADM. Following 3 hr ADM exposure, however, the SF-24 was less than the SF-0. If TFP was present during the 24 hr period after drug exposure, a considerable decrease in SF-24 compared to SF-0 was seen in both cases.

Calmodulin-induced feeding in the satiated cat: evidence for involvement of calcium and norepinephrine in the brain.

The central effect of the Ca++ binding protein, calmodulin (CaM) on spontaneous feeding as well as on core temperature was examined in the satiated cat in which chronically indwelling cannulae were permanently implanted for intracerebroventricular (ICV) infusion. When CaM was injected ICV in doses of 2.5-10.0 micrograms, the intake of food was significantly enhanced in the satiated cat without any notable change in the animal's core temperature. Ca++ ions infused similarly in a solution of 6.25-25.0 mM also augmented the spontaneous ingestion of food, which was accompanied by a concentration-related decline in core temperature. When infused separately, neither CaM in a low dose (1.25 micrograms) nor Ca++ ions (3.0 mM) given ICV altered the intake of food of the satiated cat. However, the simultaneous infusion of CaM and Ca in these concentrations enhanced significantly the amount of food consumed by as much as 60 g. When the same concentration of Ca++ ions was infused ICV simultaneously with 5.0 micrograms troponin C, a Ca++ binding protein of an identical molecular weight, the intake of food was unaltered. Further, the spontaneous feeding induced by CaM could be attenuated either by the central chelation of Ca++ ions by 1.0-1.5 mM EGTA or by 30 micrograms calcineurin, a specific CaM inhibitor, when either was given ICV. Pre-treatment of the cat with ICV phentolamine (50 micrograms) also reduced the CaM-induced feeding response significantly, whereas the similar pre-treatment with ICV propranolol (50 micrograms) or naloxone (100 micrograms) failed to affect CaM-induced feeding behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Calmodulin infused intracerebroventricularly enhances food intake in the cat.

In the fasted cat, calmodulin (CaM) infused into the cerebral ventricle produces an increase in the normal intake of food in a dose-dependent manner. The enhancement of feeding by CaM seems to be functionally specific since the response was: (1) abolished by the simultaneous intraventricular infusion of calcineurin, a specific CaM antagonist; (2) not mimicked by another calcium binding protein, troponin C; and (3) independent of the CaM's lack of effect on body temperature and water intake. This finding opens up the dual possibility that this Ca2+ binding protein may affect receptors other than intracellularly and that CaM is involved in specific functions controlled by the brain.