Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase(1).
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The deep-sea shrimp Oplophorus gracilirostris secretes a luciferase that catalyzes the oxidation of coelenterazine to emit blue light. The luciferase (M(r) approx. 106000) was found to be a complex composed of 35 kDa and 19 kDa proteins, and the cDNAs encoding these two proteins were cloned. The expression of the cDNAs in bacterial and mammalian cells indicated that the 19 kDa protein, not the 35 kDa protein, is capable of catalyzing the luminescent oxidation of coelenterazine. The primary sequence of the 35 kDa protein revealed a typical leucine-rich repeat sequence, whereas the catalytic 19 kDa protein shared no homology with any known luciferases including various imidazopyrazinone luciferases. (+info)
Lack of multicellular drug resistance observed in human ovarian and prostate carcinoma treated with the proteasome inhibitor PS-341.
(34/2047)
Almost all known conventional cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than in killing tumor cells grown as monolayer cell cultures. This "multicellular resistance" reflects the relative intrinsic drug-resistant phenotype of most solid tumors growing in vivo and is due to factors such as limited drug penetration or reduced fractions of proliferating cells. Proteasome inhibitors such as PS-341, a dipeptide boronic acid analogue, represent an interesting new class of potential anticancer drugs, which are entering early-phase clinical trials. PS-341 has been found to have good broad-spectrum cytotoxic activity in the 60-monolayer cell line National Cancer Institute screen. However, because its relative potency has not been tested in spheroid systems, we analyzed the activity of PS-341 in a spheroid/solid tumor context using four different human ovarian carcinoma cell lines and three prostate carcinoma cell lines, respectively. We found, with one exception, that PS-341 showed equal or greater activity in spheroids than in the respective monolayer cell cultures, even in a prostate cancer spheroid model with a very low growth fraction. PS-341 induced apoptotic cell death in carcinoma cells in both culture systems. We also noted a decrease in XIAP protein, a member of the inhibitor of apoptosis (IAP) family of apoptosis inhibitors, and phosphorylation of Bcl-XL in PS-341-treated ovarian carcinoma cells. Furthermore, DNA fragmentation, a hallmark of apoptosis (in this case, induced by PS-341), was completely inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). Taken together, the results indicate that unlike most other known anticancer cytotoxic drugs, PS-341 appears to be as effective in killing tumor cells grown in the form of multicell spheroids as in killing tumor cells grown in monolayer cell culture. Hence, this compound has the potential to circumvent multicellular drug resistance and, as such, may show promising activity against solid tumors with low growth fractions in vivo, which are frequently intrinsically resistant to conventional cytotoxic anticancer drugs. (+info)
Prostasomes inhibit the NADPH oxidase activity of human neutrophils.
(35/2047)
Prostasomes are particular lipid vesicles secreted by the prostate in human semen and involved in several physiological functions such as the improvement of sperm motility or immunomodulation. We have previously shown that they reduced the overall reactive oxygen species (ROS) production of seminal polymorphonuclear neutrophils (PMN). The present study was conducted to define the mechanism by which prostasomes inhibit the ROS production of blood and seminal PMN. The luminol chemiluminescence measuring total ROS production of blood PMN stimulated by either a phorbol ester (PMA) or a chemoattractant peptide, formyl-Met-Leu-Phe (fMLP) was significantly inhibited by prostasomes. The NADPH oxidase activity of the PMN was measured by 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1, 2-a]pyrazin-3-one (MCLA) chemiluminescence. Prostasomes inhibited the NADPH oxidase activity of blood or seminal PMN and increased the lag-phase of the enzyme after PMA stimulation. Prostasomes also inhibited significantly the NADPH oxidase activity of fMLP stimulated blood PMN, but the inhibition was not significant for seminal PMN. The lipid composition of blood PMN was analysed and compared to the lipid composition of prostasomes. This showed that prostasomes had a high cholesterol:phospholipid molar ratio and a high proportion of sphingomyelin. Together with the fact that prostasomes can rigidify the plasma membrane of blood PMN, these results led us to postulate that prostasomes inhibit the NADPH oxidase activity of PMN by lipid transfer from the prostasomes to the plasma membrane of the PMN. (+info)
The nicotinic acid analogue acipimox increases plasma leptin and decreases free fatty acids in type 2 diabetic patients.
(36/2047)
The effect of 3 days of intensive treatment with acipimox, an antilipolytic nicotinic acid derivative, on plasma leptin levels was studied in eight patients with Type 2 diabetes mellitus in a double-blind, placebo-controlled, cross-over study. Acipimox reduced plasma free fatty acids (FFA) markedly and lowered plasma triglycerides, glucose and insulin. Plasma leptin levels were elevated in all eight patients during 3 days of acipimox treatment (mean increase+/-s.e.: 2.38+/-0.57ng/ml, P<0.005) and the 24h mean effect of acipimox on leptin levels increased during the experimental period (P<0.03). The effect on plasma insulin and glucose resembled a mirror image of the effect on plasma leptin during 3 days of treatment. The suggestion that leptin mediates insulin resistance and may be involved in the development of the diabetic syndrome cannot be supported by the present results. It has been reported that FFA stimulates leptin secretion. Surprisingly, despite a markedly reduced FFA level, leptin concentration increased in the present study. We suggest that a primary acipimox effect is to increase leptin secretion, and that this prevails over the reduced FFA stimulus. (+info)
Endothelin receptor antagonism does not prevent the development of in vivo glyceryl trinitrate tolerance in the rat.
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There is evidence that increased endothelial production of endothelin-1 (ET-1) may contribute to glyceryl trinitrate (GTN) tolerance. We used the competitive ET(A) receptor antagonist ZD2574 to determine whether chronic ET(A) receptor blockade affected the biochemical and functional responses to GTN during the development of GTN tolerance in vivo. Tolerance induced using transdermal GTN patches resulted in a 5.3 +/- 1.2-fold increase in the EC(50) value for GTN relaxation in isolated aorta from GTN-tolerant rats. Coadministration of ZD2574 (100 mg kg(-1) t.i.d. for 3 days) during tolerance induction had no effect on GTN-induced relaxation. This dose of ZD2574 markedly blunted the pressor response to ET-1, indicating effective blockade of ET(A) receptors, and also abolished the initial transient depressor response to ET-1, indicating that blockade of endothelial ET(B) receptors also occurred using this dosage regimen for ZD2574. Consistent with the relaxation data, coadministration of ZD2574 had no effect on the decrease in GTN-induced cGMP accumulation or on the decrease in GTN biotransformation that occurred in aortae from GTN-tolerant animals. Radioimmunoassay data indicated that the GTN tolerance induction protocol caused a 2.3 +/- 0.4-fold and a 2.2 +/- 0.5-fold increase in total tissue ET-1 levels in tolerant aorta and vena cava, respectively. These data suggest that chronic inhibition of ET receptors by ZD2574 was not sufficient to prevent or diminish the tolerance-inducing effects of GTN, and that the increase in ET-1 levels observed in tolerant tissues may occur as a consequence of the vascular changes that occur during chronic GTN exposure. (+info)
Chronic daily low dose of 4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (Oltipraz) in patients with previously resected colon polyps and first degree female relatives of breast cancer patients.
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The chemoprevention agent oltipraz, one of the most active chemopreventive compounds in preclinical studies, has been shown to induce glutathione-S-transferase (GST) activity in animals. Oltipraz was evaluated in a Phase I trial at daily oral doses of 20 mg (L1), 50 mg (L2), and 100 mg (L3) and twice weekly doses of 125 mg (L4) taken for 6 months with 6 patients entered at L1 and L2 and 7 patients entered at L3 and L4 (26 subjects: 19 females and 7 males). The subject population included patients with previously resected colon polyps and first-degree female relatives of breast cancer patients. Patients with resected colon polyps underwent rectal biopsy for GST and glutathione (GSH) analyses. Of the 26 subjects, the following completed 6 months of therapy: 4 of 6 patients (L1), 4 of 6 patients (L2), 5 of 7 patients (L3), and 4 of 7 patients (L4). Toxicities were mild to severe and included: gastrointestinal symptoms, photosensitivity/heat intolerance, and neurological symptoms. Monthly plasma samples were obtained 2-3 h after oltipraz ingestion with minimally detectable plasma concentrations at L1. There was a significant difference in mean oltipraz concentration across the four doses, with no significant differences in mean oltipraz concentration over time. Rectal tissue and lymphocyte GSH and GST were variable, with no significant difference in mean levels across doses. At the 100-mg/day dose (L3), 1 patient experienced significant increase in rectal tissue GSH and GST activity, whereas 3 additional patients (L1 and L4) had >50% increase in tissue GSH. Lymphocyte GSH level was significantly related to plasma oltipraz concentration. There were no significant correlations between plasma oltipraz concentration and lymphocyte GST level nor any significant correlation between plasma concentration and percentage of change in tissue GSH or GST. Further investigation of dose/schedule and biological end points is ongoing. (+info)
In vivo modulation of vagal-identified dorsal medullary neurones by activation of different 5-Hydroxytryptamine(2) receptors in rats.
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1. In in vivo experiments, DOI (a 5-HT(2) receptor agonist), MK-212 (a 5-HT(2C) receptor agonist), and BW-723C86 (a 5-HT(2B) receptor agonist) were applied by ionophoresis to neurones in the rat nucleus tractus solitarius (NTS) receiving vagal afferent input. 2. The majority of the putative 'monosynaptically' vagal activated cells were inhibited by both MK-212 (4/6) and DOI (2/4), but unaffected by BW-723C86 (12/14). In contrast, 'polysynaptically' activated NTS cells were excited by both BW-723C86 (13/19) and DOI (9/10). Inactive 'intermediate' cells were inhibited by BW-723C86 (9/12), MK-212 (5/6) and DOI (3/4), whilst active cells of this group were excited by BW-723C86 (7/13) and DOI (5/5). 3. The selective 5-HT(2B) receptor antagonist LY-202715 significantly reduced the excitatory actions of BW-723C86 on 'intermediate' and 'polysynaptic' cells (13/13), but not the inhibitory effects observed on inactive Group 2 cells (n=5) whereas the selective 5-HT(2C) receptor antagonist RS-102221 reversed the inhibitory effects of MK-212 and DOI on 'monosynaptic and 'intermediate' neurones. 4. Cardio-pulmonary afferent stimulation inhibited two of four putative 'monosynaptically' activated calls and all four inactive intermediate cells. These were also inhibited by DOI and MK-212. In contrast, cardio-pulmonary afferents excited all five active intermediate cells and all six putative 'polysynaptically' activated NTS cells, while all were also previously excited by BW-723C86 and/or DOI. 5. In conclusion, these data demonstrate that neurones in the NTS are affected differently by 5-HT(2) receptor ligands, in regard of their vagal postsynaptic location, the type of cardio-pulmonary afferent they receive and the different 5-HT(2) receptors activated. (+info)
Stromal-derived factor 1-induced megakaryocyte migration and platelet production is dependent on matrix metalloproteinases.
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Despite the discovery of thrombopoietin (TPO) and its contribution to megakaryocytopoiesis, the exact mechanisms and sites of platelet production are unknown. It has been shown that mature megakaryocytes (MKs) functionally express the stromal-derived factor 1 (SDF-1) receptor, CXCR4. SDF-1-induced migration of mature MKs through endothelial cell layers results in increased platelet production. Because the migration of polyploid MKs from the bone marrow microenvironment requires remodeling of the perivascular extracellular matrix, it was hypothesized that mature polyploid MKs may express matrix metalloproteinases (MMPs), facilitating their exit into the bone marrow extravascular space. In this report, it is demonstrated that SDF-1 induces the expression and release of gelatinase B (MMP-9) by purified mature polyploid human MKs and an adeno-CXCR4-infected megakaryocytic cell line. Neutralizing antibody to MMP-9, but not MMP-2, blocked SDF-1-induced migration of MKs through reconstituted basement membrane, suggesting that expression of MMP-9 is critical for MK migration. Incubation of mature MKs with a synthetic MMP inhibitor, 5-phenyl-1,10-phenanthrolene, resulted in the inhibition of platelet formation, suggesting that the expression of MMPs is not only critical for megakaryocyte migration but also for subsequent platelet release. Confirming these results, adeno-SDF-1 injection into normal mice resulted in increased platelet counts, a process that could be blocked by a synthetic MMP inhibitor. These results suggest mobilization of MKs involves sequential expression and activation of chemokine receptors such as CXCR4, MMP-9, followed by transendothelial migration. MMP inhibitors may have potential use in the treatment of thrombotic and myeloproliferative disorders. (Blood. 2000;96:4152-4159) (+info)