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PubMed1994

[Limulus test (factor G pathway) positive substance during cardiopulmonary bypass].

Limulus cascade reaction is found to be frequently activated by the plasma in the patients undergoing cardiopulmonary bypass (CPB). Recently, it is considered that this phenomenon occurs by factor G pathway reactive activity (GPRA) rather than endotoxin that activates factor C pathway. In the present report, we investigated circulating endotoxin and GPRA in 32 patients undergoing CPB. Plasma was pretreated by the New PCA method, and assay was performed by two limulus tests; Toxicolor (reactive with endotoxin and GPRA) and Endospecy (endotoxin-specific). Thirty-two patients were classified into four groups by clinical diagnosis, Group A; dissecting aneurysm of the aorta (6 patients), Group B; ischemic heart disease (9 patients), Group C; heart valve disease (8 patients), Group D; congenital heart disease and myxoma in left atrium (9 patients). GPRA in Ringer's lactate hardly increased during previous circulation before operation. The highest plasma GPRA concentration in all patients was observed at the end of CPB, 415 +/- 342pg/ml, but plasma endotoxin concentration was within normal range. At the end of CPB, plasma GPRA concentrations were 761 +/- 180pg/ml in Group A, 646 +/- 341pg/ml in Group B, 230 +/- 86pg/ml in Group C, and 113 +/- 65pg/ml in Group D. GPRA increase ratio (GPRA increase value/CPB time) of Groups A and B was significantly higher (p < 0.05) than that of Groups C and D. It was suggested that the origin of plasma GPRA during CPB was not the CPB circuit but the tissue of the patients.

Adolescent↗
PubMedDate not supplied

Evaluation of a mini-cascade impactor for sampling exposure chamber atmospheres.

Andersen 4-stage mini-cascade impactors, designed primarily for personnel dosimetry, were tested for suitability of characterizing solid-particle aerosols within inhalation exposure chambers. Performances were assessed by comparing their particle-size separating capabilities against cyclone separators and Andersen 8-stage ambient cascade impactors. Data reveal excellent agreement between respirable (ACGIH) mass fractions, and mass median aerodynamic diameters, measured with the mini-samplers and the larger laboratory cascade impactors. Cyclone data did not correlate as well. Findings indicate that where accurate particle characterization at low sampling flow rates is desired, compact mini-cascade impactors are appropriate instruments.

Aerosols↗
PubMedDate not supplied

Inhibition of mitogen activated protein kinase signaling affects gastrulation and spiculogenesis in the sea urchin embryo.

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.

Animals↗
PubMed2000

Dietary regulation and localization of apoptosis cascade proteins in the colonic crypt.

This study was designed primarily to assess the localization of apoptosis cascade proteins along the rat colonic crypt and secondarily to test whether the activity and/or localization of these proteins are affected by the enrichment of the diet with the soluble fiber pectin. Expression of apoptosis cascade proteins was assessed in isolated colonocytes harvested from the luminal and basal crypt colonocyte populations. Two different dietary regimens were tested: a standard diet (diet A), and a diet enriched in pectin (diet B), a soluble fiber that undergoes fermentation in the cecum and produces high concentrations of intracolonic short-chain fatty acids. Caspase-1 expression was maximal in luminal colonocytes of rats fed diet B, as evidenced by Western blot and immunohistological analyses. Expression of the cleaved poly(ADP-ribose) polymerase product was elevated in both the luminal and basal colonocytes of the pectin-fed group, whereas in rats fed diet A, the expression was lower, especially in basal crypt colonocytes. The highest expression of the antiapoptotic protein Bcl-2 was observed in the lower compartments of the colonic crypt tissue and was maximal in the rat group fed a standard diet. The apoptotic index in colonocytes of rats fed diet B was higher than that measured in rats fed diet A. Cumulatively, our results indicate that apoptosis cascade proteins are differentially localized along the lumen-crypt axis, and their expression and activity may be controlled by dietary components. These results may, at least partially, account for the documented protective effect of butyrogenic fibers on colorectal cancer.

Animals↗
PubMed2001

K+ stimulates specifically the autokinase activity of purified and reconstituted EnvZ of Escherichia coli.

The histidine kinase/response regulator system EnvZ/OmpR of Escherichia coli regulates transcription of the genes ompF and ompC, encoding two porins of the outer membrane. Although the total amount of OmpF and OmpC remains constant, the relative levels of the two proteins fluctuate in a reciprocal manner depending on medium osmolality. The membrane-anchored sensor EnvZ somehow monitors changes in environmental osmolality. To characterize the nature of the stimulus perceived by EnvZ, this protein was overproduced, purified, and reconstituted into proteoliposomes. Autokinase activity of purified and reconstituted EnvZ was stimulated by an increase of the K(+) concentration. Rb(+), Na(+), and NH4(+) also stimulated the activity but to a smaller extent, whereas an osmotic upshift imposed by various sugars or increasing concentrations of glycine betaine, proline, or Tris/MES were without influence. Neither the transfer of the phosphoryl group from EnvZ approximately P to OmpR nor the EnvZ-mediated OmpR approximately P dephosphorylation were affected by one of the tested solutes. Experiments with the reconstructed signal transduction cascade including DNA fragments demonstrated a substantial increase of the amount of phosphorylated OmpR in the presence of K(+) and to a lower extent in the presence of Na(+), Rb(+), and NH4(+). Various K(+) salts were tested indicating that the determined effects were K(+)-specific and not dependent on the anion. In a further in vitro test system, which utilizes right-side-out membrane vesicles, the K(+)-specific activation of EnvZ autokinase from the luminal side was confirmed. These results clearly indicate a regulation of EnvZ autokinase activity by monovalent ions, specifically K(+). Whether K(+) accumulation, which is one of the first responses of E. coli after an osmotic upshift, is related to the stimulation of the EnvZ autokinase activity in vivo is discussed.

Bacterial Outer Membrane Proteins↗
PubMed2003

Effects of strenuous exercise on haemostasis.

OBJECTIVES: To review the effects of exercise on haemostasis and examine the possible clinical sequelae of these changes. METHODS: The search strategy included articles from 1966 to August 2002 using Medline and SportDiscus databases, and cross referencing the bibliographies of relevant papers. RESULTS: Exercise results in activation of both the coagulation and fibrinolytic cascades, as shown by a reduction in whole blood clotting time and activated partial thromboplastin time, an increase in the activity of several components of the cascades, and an increase in fibrin degradation products. In vitro tests suggest that coagulation remains activated after fibrinolysis has returned to baseline levels. CONCLUSIONS: Both the coagulation and fibrinolytic cascades are stimulated by strenuous exercise, but the temporal relation between the two and its clinical significance remains to be clarified. Doctors and athletes should be aware of the haemostatic changes induced by exercise, and further work is needed to clarify the possible role of these changes in sudden cardiac death.

Blood Coagulation↗
PubMed2000

Differential response to transforming growth factor (TGF)-alpha and fibroblast growth factor (FGF) in human renal cell carcinomas of the clear cell and papillary types.

The clear cell and the papillary types of human renal cell carcinoma (RCC) are distinct tumour entities with marked differences in their biological properties. Because growth factors are considered to affect profoundly the biological behaviour of malignant tumours, we compared the expression and function of transforming growth factor (TGF)-alpha and fibroblast growth factor (FGF) in both types of RCCs. Both in vivo and in vitro expression of TGF-alpha, epidermal growth factor-receptor (EGF-R), FGF-2 and FGF type 3- and 4-receptors was found in RCCs of both types. However, marked differences between clear cell and papillary RCCs became evident for TGF-alpha secretion, which could be demonstrated in 20 out of 24 (83%) clear cell RCCs but in only two out of four (50%) papillary tumours. Moreover, the mean TGF-alpha secretion rate in clear cell RCCs significantly (P<0. 05) exceeded that of papillary RCCs. Because the expression of growth factor receptors could not prove the corresponding signalling cascades were functional, tumour cell proliferation was tested after exposure to exogenous TGF-alpha or FGF-1. These experiments demonstrated that papillary RCCs did not respond significantly to exogenous TGF-alpha or FGF-1, whereas eight (33%) (TGF-alpha) and 11 (46%) (FGF-1) out of 24 clear cell RCCs responded with significant (P<0.05) growth stimulation. In conclusion, our investigation presents data indicating that TGF-alpha and FGF are functionally involved in the progression of clear cell RCCs, directly stimulating proliferation by autocrine and/or paracrine actions. In contrast, TGF-alpha and FGF did not directly stimulate the proliferation of our papillary RCCs, thereby suggesting functional defects or a blockade in the corresponding signalling cascades. This differential functionality might contribute to the more aggressive behaviour of clear cell RCCs.

Adenocarcinoma, Clear Cell↗
PubMed1993

PKC-mediated redistribution of mitogen-activated protein kinase during smooth muscle cell activation.

Protein kinase C (PKC) translocates from the cytosol to the surface membrane at the time it mediates agonist-induced contraction of ferret vascular smooth muscle cells (R. A. Khalil and K. G. Morgan. J. Physiol. Lond. 455: 585-599, 1992). However, no direct communication between membrane-associated PKC and the contractile filaments has been identified. Mitogen-activated protein (MAP) kinase is a substrate for PKC and is also capable of phosphorylating the actin-binding protein caldesmon at sites phosphorylated during smooth muscle contraction in vivo (L. P. Adam, C. J. Gapinski, and D. R. Hathaway. FEBS Lett. 302: 223-226, 1992). In the present study, the hypothesis that PKC and MAP kinase are involved in a signal-transduction cascade leading to smooth muscle contraction was tested. Immunofluorescence and digital-imaging microscopy were used to localize the epsilon-PKC isoform and MAP kinase during phenylephrine-induced Ca(2+)-independent activation of ferret aorta cells. We report that maintained phenylephrine-induced translocation of cytosolic PKC to the surface membrane is associated with transient redistribution of cytosolic MAP kinase to the surface membrane before cell contraction. Coincident with cell contraction, MAP kinase undergoes a second redistribution away from the plasmalemma and toward the vicinity of contractile filaments. Redistribution of MAP kinase is not stimulated by Ca2+ but is completely prevented by PKC inhibitors. The transient Ca(2+)-independent but PKC-dependent redistribution of MAP kinase points to MAP kinase as a missing link in the signal-transduction cascade between membrane-bound PKC and smooth muscle activation.

Animals↗
PubMed1991

Modulation of the human sperm acrosome reaction by effectors of the adenylate cyclase/cyclic AMP second-messenger pathway.

The acrosome reaction of spermatozoa appears to be analogous to various somatic cell exocytotic events which involve cascade reactions, i.e., transmission of an external signal across the cell membrane resulting in activation of an "amplifier" enzyme and the generation of a second messenger. Using a synchronous acrosome reaction system (De Jonge et al., J. Androl., 10:232-239, '89a), it was found that analogues of the second-messenger cAMP, dibutyryl cAMP (dbcAMP) and 8-bromo cAMP, stimulated the acrosome reaction of capacitated spermatozoa. Additionally, treatment of spermatozoa with either xanthine or non-xanthine phosphodiesterase inhibitors induced a significant (P less than 0.05) increase in the percent acrosome reaction after a period of capacitation in comparison to untreated controls. These results indicate that analogues of cAMP or inhibitors which prevent cAMP hydrolysis can induce the human sperm acrosome reaction. Subsequent experiments were conducted to test whether the amplifier enzyme in the cascade reaction, adenylate cyclase, has a role in the acrosome reaction. Forskolin, an adenylate cyclase stimulator, caused a significant (P less than 0.01) increase in the percent acrosome reaction in comparison to controls. Modulators of adenylate cyclase--adenosine, 2'-0-methyladenosine, and 2',3'-dideoxyadenosine--significantly (P less than 0.01) inhibited the forskolin-induced acrosome reaction. dbcAMP was able to overcome the inhibition by adenosine. Two inhibitors of protein kinase A, the Walsh inhibitor and H-8, caused a significant (P less than 0.01) inhibition of the dbcAMP-induced acrosome reaction. Finally, in the absence of extracellular calcium, dbcAMP induced a significant (P less than 0.01) increase in the acrosome reaction in contrast to A23187. These results suggest that: 1) a molecular mechanism for the human sperm acrosome reaction involves the cAMP second-messenger system; i.e., activation of adenylate cyclase, the amplifier enzyme that produces cAMP, production of cAMP as a second messenger, and activation of cAMP-dependent kinase A; and that 2) activation of adenylate cyclase occurs after calcium influx.

Acrosome↗
PubMed2001

Validation of laser diffraction method as a substitute for cascade impaction in the European Project for a Nebulizer Standard.

The project for a European standard testing procedure to characterize nebulizers in terms of particle size distribution has been based on using the Andersen-Marple personal cascade impactor model 298 (A-MPCI) with a sodium fluoride reference solution. In the present study methods based on laser diffraction (Mastersizer-X) and time-of-flight (TOF)(APS) and another cascade impactor (GS1-CI) were compared with the A-MPCI. Two types of nebulizer (Pari LC+ and Microneb) were tested with all apparatuses, and a third type of nebulizer (NL9) was tested with the A-MPCI and Mastersizer-X. Nebulizers were charged with a solution of sodium fluoride in conditions reproducing the European Committee for Normalization (CEN) protocol. There was no difference between the Mastersizer-X and the A-MPCI or between the GS1-CI and the A-MPCI in terms of mass median aerodynamic diameter (MMAD). Comparison between the APS and the A-MPCI showed a significant difference with the Microneb. The geometric standard deviations (GSD) obtained with the A-MPCI were on average 10% greater than GSD obtained with the other apparatuses, but the differences were not statistically significant. We conclude that laser diffraction can be used for particle size distribution in the context of the European standard, and that the Mastersizer-X is particularly interesting for industrial practice in view of its simplicity and robustness.

Equipment Design↗
PubMed1996

Rabbit complement lyses tumor cells without massive C3 deposition.

These experiments were performed to determine why rabbit complement lyses tumor cells very efficiently, while not having particularly strong activity in hemolytic assays or in any other complement assay. The target cells used were human tumor cells coated with three different mouse IgG(2a) monoclonal antibodies, and complement from 5 mammalian species were tested. In antibody titration experiments, rabbit complement was found to lyse target cells at a relatively low antibody concentration, insufficient to allow lysis by complement of other species. Since this result was still observed after absorption of rabbit serum with target cells, the potency of rabbit complement cannot be attributed to the presence of natural antibodies. We then assayed C3 deposition on target cells, using two types of (125)I-labeled anti-C3 Abs to measure C3 deposition: goat antibodies specific for C3 of the human, guinea pig, rabbit, rat or mouse, and chicken antibodies to human C3 which cross-react with C3 of other mammals. Unexpectedly, complement of the human, rat, guinea pig, and BUB mouse deposited large amounts of C3 on the surface of target cells, while rabbit complement deposited 100-1,000 fold less. We discuss the possible reasons that C3 deposition does not correlate with cytotoxicity, and may indeed be inversely related. These data indicate that there is a fundamental difference in the complement cascade between rabbits and the other species tested. The potent lytic activity of rabbit complement is likely to be related to this difference, although the mechanism is not yet understood.

Animals↗
PubMed2006

Imaging of cAMP levels and protein kinase A activity reveals that retinal waves drive oscillations in second-messenger cascades.

Recent evidence demonstrates that low-frequency oscillations of intracellular calcium on timescales of seconds to minutes drive distinct aspects of neuronal development, but the mechanisms by which these calcium transients are coupled to signaling cascades are not well understood. Here we test the hypothesis that spontaneous electrical activity activates protein kinase A (PKA). We use live-cell indicators to observe spontaneous and evoked changes in cAMP levels and PKA activity in developing retinal neurons. Expression of cAMP and PKA indicators in neonatal rat retinal explants reveals spontaneous oscillations in PKA activity that are temporally correlated with spontaneous depolarizations associated with retinal waves. In response to short applications of forskolin, dopamine, or high-potassium concentration, we image an increase in cAMP levels and PKA activity, indicating that this second-messenger pathway can be activated quickly by neural activity. Depolarization-evoked increases in PKA activity were blocked by the removal of extracellular calcium, indicating that they are mediated by a calcium-dependent mechanism. These findings demonstrate for the first time that spontaneous activity in developing circuits is correlated with activation of the cAMP/PKA pathway and that PKA activity is turned on and off on the timescale of tens of seconds. These results show a link between neural activity and an intracellular biochemical cascade associated with plasticity, axon guidance, and neural differentiation.

Animals↗
PubMed1994

Fibrin formation and degradation in patients with arteriosclerotic disease.

BACKGROUND: The blood coagulation cascade was reported to be activated in patients with arteriosclerotic disease of the lower limbs (peripheral arterial disease, PAD). There is more thrombin and fibrin formation compared with healthy control subjects. In many studies, however, the presence of arteriosclerotic disease had not been thoroughly ruled out in the control group. Therefore, markers of the activation of the blood coagulation cascade were measured in patients with PAD and in a carefully defined control group, both groups being subjected to an exercise test. METHODS AND RESULTS: Twenty-two patients with angiographically documented PAD of grade II (Fontaine classification) and 13 control subjects in whom the presence of arteriosclerotic lesions was ruled out by noninvasive means in the carotid arteries, abdominal aorta, leg arteries, and coronary arteries took part in the study. Before and immediately after a treadmill stress test, the concentrations of prothrombin fragment F1 + 2 (F1 + 2), thrombin-antithrombin III complexes (TAT), fibrinopeptide A (FPA; this peptide was measured in spot urine also), and D-dimers were measured. Before exercise, the concentrations of F1 + 2 (1.0 +/- 0.6 versus 0.7 +/- 0.3 nmol/L), TAT (2.9 +/- 2.1 versus 1.9 +/- 0.8 micrograms/L), and D-dimers (318.2 +/- 270.1 versus 150.0 +/- 91.4 micrograms/L) were significantly higher in the patients with PAD compared with the healthy control subjects. FPA concentrations in plasma (1.9 +/- 1.0 versus 1.4 +/- 0.6 micrograms/L) and spot urine were not different, however. F1 + 2, FPA, and D-dimer concentrations correlated with the severity of the PAD as assessed by the ankle systolic blood pressure index (ABPI). The symptom-limited stress test did not lead to further activation of the blood coagulation cascade. However, concentrations of F1 + 2 (P < .001) and TAT (P < .01) after exercise correlated with the presence of ischemic changes in the stress-test ECG. CONCLUSIONS: There is evidence of enhanced thrombin formation in patients with PAD compared with an age- and sex-matched control group without clinical and sonographic evidence of arteriosclerosis. The thrombin formed, however, appears to be almost completely neutralized by antithrombin III. No direct evidence of fibrin formation was obtained, since the FPA concentrations were not different. In the patients with PAD, the higher concentrations of D-dimers are indicative of in vivo fibrinolysis. Thus, some fibrin formation must be postulated to occur in patients with arteriosclerosis.

Aged↗
PubMed2003

Role of thalamic phospholipase C[beta]4 mediated by metabotropic glutamate receptor type 1 in inflammatory pain.

Phospholipase C (PLC) beta4, one of the four isoforms of PLCbetas, is the sole isoform expressed in the mouse ventral posterolateral thalamic nucleus (VPL), a key station in pain processing. The mouse thalamus also has been shown to express a high level of metabotropic glutamate receptor type 1 (mGluR1), which stimulates PLCbetas through activation of Galphaq/11 protein. It is therefore expected that the thalamic mGluR1-PLCbeta4 cascade may play a functional role in nociceptive transmission. To test this hypothesis, we first studied behavioral responses to various nociceptive stimuli in PLCbeta4 knock-out mice. We performed the formalin test and found no difference in the pain behavior in the first phase of the formalin test, which is attributed to acute nociception, between PLCbeta4 knock-out and wild-type mice. Consistent with this result, acute pain responses in the hot plate and tail flick tests were also unaffected in the PLCbeta4 knock-out mice. However, the nociceptive behavior in the second phase of the formalin test, resulting from the tissue inflammation, was attenuated in PLCbeta4 knock-out mice. In the dorsal horn of the spinal cord where PLCbeta1 and PLCbeta4 mRNAs are expressed, no difference was found between the wild-type and knock-out mice in the number of Fos-like immunoreactive neurons, which represent neuronal activity in the second phase in the formalin test. Thus, it is unlikely that spinal PLCbeta4 is involved in the formalin-induced inflammatory pain. Next, we found that pretreatment with PLC inhibitors, mGluR1 antagonists, or both, by either intracerebroventricular or intrathalamic injection, attenuated the formalin-induced pain behavior in the second phase in wild-type mice. Furthermore, activation of mGluR1 at the VPL enhanced pain behavior in the second phase in the wild-type mice. In contrast, PLCbeta4 knock-out mice did not show such enhancement, indicating that mGluR1 is connected to PLCbeta4 in the VPL. Finally, in parallel with the behavioral results, we showed in an electrophysiological study that the time course of firing discharges in VPL corresponds well to that of pain behavior in the formalin test in both wild-type and PLCbeta4 knock-out mice. These findings indicate that the thalamic mGluR1-PLCbeta4 cascade is indispensable for the formalin-induced inflammatory pain by regulating the response of VPL neurons.

Action Potentials↗
PubMed2005

Caveolae modulate excitation-contraction coupling and beta2-adrenergic signalling in adult rat ventricular myocytes.

OBJECTIVE: Caveolae, flask shaped invaginations of the cell membrane, influence signalling cascades in many cell types. We have tested the hypothesis that caveolae modulate excitation-contraction coupling (ECC) and beta-adrenergic stimulation in the adult cardiac myocyte. METHODS: Shortening, [Ca(2+)](i) and L-type Ca(2+) current (I(Ca,L)) were recorded in rat ventricular myocytes. Caveolae were disrupted with methyl-beta-cyclodextrin (MbetaC). RESULTS: Shortening and [Ca(2+)](i) transient amplitude were reduced in myocytes treated with MbetaC. MbetaC did not alter the density or characteristics of I(Ca,L) or the sarcoplasmic reticulum (SR) Ca(2+) load, but significantly reduced fractional SR Ca(2+) release. The inotropic response of myocytes to beta(1)-adrenoceptor stimulation was insensitive to caveolae disruption. By contrast, the increase in shortening, [Ca(2+)](i) transient and I(Ca,L) seen following beta(2) stimulation was markedly enhanced (3-5 fold) following MbetaC treatment, and the effect on I(Ca,L) could be mimicked by dialyzing cells with an antibody to caveolin 3. When the G(alphai) pathway was disabled with pertussis toxin (PTX), control cells showed a similar response to beta(2) stimulation as seen in MbetaC-treated myocytes, whereas MbetaC-treated cells were insensitive to PTX. CONCLUSIONS: Caveolae modulate ECC via the efficiency of the Ca(2+)-induced Ca(2+) release process, rather than Ca(2+) influx. Our data are also consistent with the hypothesis that interaction of G(i) protein cascade components with caveolin in the caveolae is necessary for effective signalling by this pathway. This suggests that changes in caveolin expression in the adult heart seen during aging and in disease will have consequences for baseline cardiac function and beta-adrenergic responsiveness.

Adrenergic beta-Antagonists↗
PubMed2004

Signal transduction pathways in Burkitt's lymphoma cell lines BL41 and DG75 with different sensitivity to doxorubicin.

AIM: To understand the biochemical basis of cell sensitivity to cytotoxic effect of doxorubicine (DOX), we investigated signaling cascades mediated by c-Jun N-terminal protein kinases (JNK1/2), p38 mitogen-activated protein kinases (MAPK), extracellular signal-regulated protein kinase (ERK1/2) and protein kinase B/Akt in both DOX-sensitive BL41 and the DOX-resistant DG75 Burkitt's lymphoma (BL) cell lines. METHODS: To test the effect of DOX on different signaling cascades, BL41 and DG75 cells were treated with DOX for varying lengths of time. Cytotoxic effect of DOX was analyzed by Hoechst 33342 staining. Total amount of JNK1/2, ERK1/2, p38 MARK, Akt proteins, and also phosphorylated/activated forms of these enzymes were detected using Western blot analysis with specific antibodies. Immunophenotypic analysis of BL41 and DG75 cells was performed by indirect immunofluorescence staining. RESULTS: Our findings demonstrated that DOX treatment of the BL41 cells led to sustained activation of JNK1/2 and p38 MAPK. This activation/phosphorylation did not result from increased expression of either JNK1/2 or p38 MAPK since protein levels of JNK1/2 and p38 MAPK in DOX-treated and untreated cells were unaltered. Apoptotic signaling cascade induced by DOX in BL41 cell was accompanied by Akt dephosphorylation. The effect of DOX in drug-resistant cell line DG75 convoyed by dephosphorylation of JNK1/2, p38 MAPK and activation of Akt. Fate of BL cells did not depend from ERK activity. CONCLUSION: The outcome of cellular response to DOX in BL cell lines is determined by interference of at least three signaling pathways: JNK1/2, p38 MAPK and PKB/Akt. The balance between Akt/PKB and MAPK pathways is important in determining whether BL cells survive or undergo apoptosis in response to DOX treatment.

Antibiotics, Antineoplastic↗
PubMed2006

Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c.

BACKGROUND: Histamine is known to elicit a negative feedback effect on anti-IgE and allergen-induced basophil activation. A series of experiments performed between 1981 and 1995 using a manual method showed biological activity of highly diluted histamine. Most of the experiments used histamine in the range 10(-30) (15C)-10(-36) M (18C). These results were confirmed by automated flow cytometry, but this method is based on the selection of basophils by anti-IgE and analysis of basophil activation by anti-CD 63, showing significant but relatively low inhibition (approximately 14%), insufficient to convince the scientific community of the reality of the phenomenon. OBJECTIVE: We investigated if the use of CD 203c, a basophil specific, earlier marker than CD 63 of the activation cascade, increased the sensitivity of the method, testing two target histamine dilutions, 10(-4) (2C) and 10(-32) M (16C). METHODS: Basophils, obtained from buffy coats, were pre-incubated with the histamine dilutions and activated by two agonists: anti-IgE and fMLP (formyl-methionyl-leucyl-phenylalanine peptide). Basophil activation was stopped with EDTA. The cells were labelled with anti-IgE, anti-CD 13 and anti-CD 14 for basophil selection, and anti-CD 63 and anti-CD 203c for basophil activation. Results were expressed in up-regulation percentage for CD 63 or mean intensity of fluorescence (MFI) for CD 203c. RESULTS: Histamine 10(-4) M (2C) and histamine 10(-32) M (16C) were capable of inhibiting both IgE-dependent (anti-IgE) and IgE-independent (fMLP) basophil activation. The percentage inhibition depended on the activation marker used. The highest inhibition for histamine dilution 16C was observed with CD 203c (38%, P<0.001), approximately half the inhibition observed with histamine 2C (73%). CONCLUSION: These new flow cytometric protocols confirmed that high dilutions of histamine may inhibit basophil activation and that the inhibitory effect is not restricted to IgE-dependent activation. The use of CD 203c instead of CD 63 increased the magnitude of the response.

Antibodies, Anti-Idiotypic↗
PubMed2006

Co-deposition of salmeterol and fluticasone propionate by a combination inhaler.

The combination of the long-acting beta2-agonist, salmeterol xinafoate (salmeterol) and inhaled corticosteroid, fluticasone propionate (FP) (Seretide/Advair) has shown enhanced efficacy compared with concurrent administration of the two drugs from individual inhalers at the same dose. A possible explanation for this increased effect is a higher degree of co-deposition of the two drugs from the combination (Seretide) inhaler compared with the component drugs administered separately. Raman laser spectroscopy, a technique capable of identifying individual drug particles, has been used with novel statistical methodology that we have developed, to determine whether there is any co-association between drug particles and whether this occurs in the Seretide formulation rather than by chance. Samples from a combined Seretide metered dose inhaler (MDI, 25/50 mcg) and salmeterol (25 mcg) with FP (50 mcg) from separate MDI's taken from Plate 4 of an Anderson Cascade Impactor were analysed. Using a statistical test based on the bootstrap technique, it was found that the co-deposition of FP and salmeterol particles from the combination MDI was significantly greater than from the separate inhalers group (p < 0.001). A higher degree of co-deposition on the same cells of the airways may possibly account for the increased efficacy observed in patients prescribed Seretide MDI.

Adrenergic beta-Agonists↗