ABSTRACT
Oral squamous cell carcinoma (OSCC) frequently metastasizes to cervical lymph nodes, which is the most known prognostic factor. Screening methods to identify sentinel lymph nodes (SLNs) are therefore of great interest for the management of potential neck metastasis. The purpose of this study was to evaluate the clinical benefit of double SLN mapping with indocyanine green (ICG) and 99m-technetium-tin colloid ((99m)Tc-tin colloid) for sentinel node navigation surgery (SNNS). Between 2007 and 2010, 16 patients diagnosed with OSCC were investigated by SLN biopsy using the double mapping method. (99m)Tc-tin colloid was injected into the peri-tumoural region on the preoperative day, and ICG was administered intraoperatively in the same position to assist in detecting nodes during surgery. Based on the gamma-ray signal and near-infrared (NIR) fluorescence of ICG, SLNs were identified and thereafter assessed pathologically and genetically for cancer involvement. Radio-guided detection was successful for all patients. ICG mapping identified a relatively larger number of nodes, suggesting that several non-SLNs were potentially involved. The double mapping method assisted surgeons to explore SLNs. Since the ICG fluorescence was shielded by the subcutaneous fatty tissue and the muscle layer including platysma and sternocleidomastoid, it was necessary to retract the tissue away from nodes.
Subject(s)
Carcinoma, Squamous Cell/diagnostic imaging , Lymphoscintigraphy , Mouth Neoplasms/diagnostic imaging , Sentinel Lymph Node Biopsy/methods , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/surgery , Coloring Agents , Female , Fluorescence , Humans , Indocyanine Green , Lymphatic Metastasis , Male , Middle Aged , Mouth Neoplasms/pathology , Mouth Neoplasms/surgery , Prognosis , Survival Rate , Technetium Compounds , Tin CompoundsABSTRACT
BACKGROUND: Propofol is an alternative to thiopental for induction of general anaesthesia for cesarean section. It crosses the placenta and induces vasodilatation of isolated vessels and may therefore alter fetal placental vascular resistance. The direct effect of propofol on the fetal placental circulation was studied in vitro. The actions of propofol on vasoconstrictive effects induced by angiotensin II (Ang II), bradykinin (BK), prostaglandin F(2alpha) (PGF(2alpha)) and potassium chloride (KCl) were evaluated. METHODS: Full-term healthy human placentas (n=48) were perfused with modified Tyrode's solution using a pulsatile pump. Placental perfusion pressure was measured in response to injection of Ang II, BK, KCl and PGF(2alpha) before and after perfusion with propofol (1.7 x 10(-5) and 5.6 x 10(-5) M). RESULTS: BK, Ang II, KCl and PGF(2alpha) induced a dose-dependent increase in placental perfusion pressure. Propofol induced a concentration-dependent decrease in placental perfusion pressure, but this was not observed with the propofol solvent (Intralipid). Propofol, but not Intralipid, reduced the vasoconstrictor effects of BK, KCl and PGF(2alpha), while the effect of Ang II was not changed. The effect of KCl was abolished in placentas perfused with Ca(2+)-free solution, while the effect of Ang II was not altered. CONCLUSIONS: Propofol induced vasodilatation and inhibited the vasoconstrictive effects of BK and PGF(2alpha), in the human placenta. These findings suggest that propofol may not reduce fetal placental blood flow. Since propofol reduced the vasoconstricting effect of KCl but not that of AngII, we propose that the vasodilatory effect of propofol in the human placenta involves inhibition of Ca(2+) channels.
Subject(s)
Anesthetics, Intravenous/adverse effects , Fetus/blood supply , Placenta/blood supply , Propofol/adverse effects , Adult , Angiotensin II/pharmacology , Blood Pressure/drug effects , Bradykinin/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Dinoprost/pharmacology , Dose-Response Relationship, Drug , Female , Humans , In Vitro Techniques , Potassium Chloride/pharmacology , Pregnancy , Pulmonary Circulation/drug effects , Regional Blood Flow/drug effects , Vasoconstriction/drug effectsABSTRACT
1. The vasodilator action of angiotensin (Ang) II has not yet been demonstrated in spontaneously hypertensive rats (SHR), nor have any possible changes in this response during the development of hypertension. 2. In the present study, the vasodilator effect of AngII was evaluated in the rat isolated, preconstricted mesenteric arterial bed (MAB) from 6- (young) and 24-week-old (adult) SHR and compared with effects on MAB from age-matched normotensive rats (control). 3. Angiotensin II (10-300 nmol) induced vasodilation in noradrenaline (NA)-preconstricted MAB that was greater in vessels from young compared with adult rats in both the control and SHR groups. Angiotensin II-induced vasodilation was reduced by the angiotensin AT(2) receptor antagonist PD 123319 (10 micromol/L), the angiotensin-(1-7) receptor antagonist A779 (1 micromol/L) and the bradykinin B(2) receptor antagonist HOE-140 (0.01 micromol/L), but not by the AT(1) receptor antagonist losartan (30 micromol/L). Expression of AT(2) receptors was weak in vessels from adult control rats compared with that in young control rats, whereas in young SHR AT(2) receptor expression was increased compared with that in young control rats. This increased expression of AT(2) receptors was maintained in adult SHR and there was no significant difference in AT(2) receptor expression between young and old SHR. 4. The findings of the present suggest that AngII induces an AT(2) receptor-mediated vasodilator effect in the MAB via activation of angiotensin-(1-7) and bradykinin receptors, an action that is reduced in adult control rats and adult SHR. In adult control rats, the attenuated response of AngII is probably due to endothelial dysfunction and reduced expression of AT(2) receptors, whereas in adult SHR it is associated with endothelial dysfunction alone. Increased expression of AT(2) receptors in SHR may represent a counteracting response for modulating blood pressure.
Subject(s)
Angiotensin II/metabolism , Gene Expression Regulation/physiology , Receptor, Angiotensin, Type 2/metabolism , Vasodilation/physiology , Acetylcholine/pharmacology , Aging , Angiotensin II/analogs & derivatives , Angiotensin II/pharmacology , Animals , Blotting, Western , Bradykinin/analogs & derivatives , Bradykinin/pharmacology , Imidazoles/pharmacology , Losartan/pharmacology , Mesenteric Arteries/drug effects , Mesenteric Arteries/metabolism , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/metabolism , Peptide Fragments/pharmacology , Pyridines/pharmacology , Rats , Rats, Inbred SHR , Receptor, Angiotensin, Type 2/geneticsABSTRACT
The mechanisms involved in the vasodilation action of clonidine have not yet been completely elucidated. We investigated the potential mechanisms that seem to be involved in the clonidine vasodilator effect using rat isolated mesenteric arterial bed (MAB). In precontracted MAB, clonidine (10-300 pmol) induced a dose-dependent relaxation, that was inhibited by endothelium removal (deoxycholic acid - 2.5 mM) and reduced by the alpha(2) adrenoceptor inhibitors yohimbine (1-3 microM) and rauwolscine (1 microM). The endothelium-dependent vasodilation induced by clonidine was reduced by the nitric oxide (NO) synthase inhibitor L-NAME (0.3 mM) and guanylyl cyclase inhibitor ODQ (10 microM) but was not affected by indomethacin (3-10 microM) alone. High K+ (25 mM) solution reduced the vasodilator effect of clonidine that was further attenuated by L-NAME. In the presence of high K+ plus L-NAME, the residual vasodilator effect of clonidine was further reduced by indomethacin (3 microM). The Ca(2+)-dependent K+ channel (K+(Ca2+)) inhibitors, charybdotoxin (ChTx; 0.1 microM) plus apamin (0.1 microM), also reduced the vasodilation induced by clonidine, however this response was not further reduced in the presence of L-NAME as observed with acetylcholine (10 pmol). In the presence of ATP-dependent K+ channel (K+(ATP)) blocker, glibenclamide (10 microM), the inhibitory effect of ChTx plus apamin plus L-NAME was increased. In contrast, the vasodilation induced by clonidine was not affected by voltage-dependent K+ channels (K(V)) blocker, 4-aminopyridine (4-AP, 1 mM). In conclusion, our results demonstrate that clonidine activates alpha(2)-adrenoceptors in rat MAB and that the endothelium-dependent vasodilation is mediated by activation of NO-cGMP pathway, hyperpolarization due to activation of K+(Ca) and K+(ATP) channels. Prostaglandins might participate in the vasodilator effect of clonidine when NO and EDHF mechanisms are blunted.
Subject(s)
Adrenergic alpha-Agonists/pharmacology , Clonidine/pharmacology , Cyclic GMP/metabolism , Mesenteric Arteries/drug effects , Nitric Oxide/metabolism , Potassium Channels/drug effects , Vasodilation/drug effects , Vasodilator Agents/pharmacology , Adrenergic alpha-Antagonists/pharmacology , Animals , Biological Factors/metabolism , Dose-Response Relationship, Drug , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Enzyme Inhibitors/pharmacology , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/metabolism , In Vitro Techniques , Male , Mesenteric Arteries/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Potassium/metabolism , Potassium Channel Blockers/pharmacology , Potassium Channels/metabolism , Potassium Channels, Calcium-Activated/drug effects , Potassium Channels, Calcium-Activated/metabolism , Prostaglandins/metabolism , Rats , Rats, Wistar , Receptors, Adrenergic, alpha-2/drug effects , Receptors, Adrenergic, alpha-2/metabolismABSTRACT
Açai (Euterpe oleracea Mart.) a fruit from the Amazon region, largely consumed in Brazil is rich in polyphenols. Experiments were undertaken to determine whether hydro-alcoholic extract obtained from stone of açaí induces a vasodilator effect in the rat mesenteric vascular bed precontracted with norepinephrine (NE) and, if so, to elucidate the underlying mechanism. Açai stone extract (ASE, 0.3-100 microg) induced a long-lasting endothelium-dependent vasodilation that was significantly reduced by N(G)-nitro-l-arginine methyl ester (l-NAME) and (1)H-[1,2,3] oxadiazolo [4,4-a] quinoxalin-l-one (ODQ) and abolished by KCl (45 mM) plus l-NAME. In vessels precontrated with NE and KCl (45 mM) or treated with K(Ca)(+2) channel blockers (charybdotoxin plus apamin), the effect of ASE was significantly reduced. However this effect is not affect by indomethacin, glybenclamide and 4-aminopiridine. Atropine, pyrilamine, yohimbine and HOE 140 significantly reduced the vasodilator effect of acetylcholine, histamine, clonidine and bradykinin, respectively, but did not change the vasodilator effect of ASE. In cultured endothelial cells ASE (100 microg/mL) induced the formation of NO that was reduced by N(G)-nitro-l-arginine (l-NA, 100 microM). The present study demonstrates that the vasodilator effect of ASE is dependent on activation of NO-cGMP pathway and may also involve endothelium-derived hyperpolarizing factor (EDHF) release. The vasodilator effect suggest a possibility to use ASE as a medicinal plant, in the treatment of cardiovascular diseases.
Subject(s)
Arecaceae , Endothelium, Vascular/drug effects , Mesenteric Arteries/drug effects , Vasodilation/drug effects , Vasodilator Agents/pharmacology , Animals , Arecaceae/chemistry , Biological Factors/metabolism , Brazil , Cells, Cultured , Cyclic GMP/metabolism , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelium, Vascular/metabolism , Endothelium-Dependent Relaxing Factors/metabolism , Fruit , Guanylate Cyclase/metabolism , Male , Mesentery/blood supply , Nitric Oxide/metabolism , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Plants, Medicinal , Potassium Channels/drug effects , Rats , Rats, Wistar , Time Factors , Vasoconstrictor Agents/pharmacology , Vasodilator Agents/isolation & purificationABSTRACT
Environmental pollution by low concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) is a concern these days due to ever increasingly stringent regulations. Photocatalysis with immobilized TiO2 fiber is a promising oxidation method. Laboratory experiments on photocatalytic degradation of 0.045 mmol l(-1) 2,4-D with the world's first high-strength TiO2 fiber catalyst were carried out in a continuous flow reactor in which the degradations were, in general, similar to those with high 2,4-D concentrations investigated elsewhere. Degradation and mineralization of 2,4-D were significantly enhanced with no initial pH adjustments. The rate constants for total organic carbon (TOC) without pH adjustment were about two-fold bigger than the pH adjustment cases. CO2 gas measurement and carbon mass-balance were carried out for the first time, where about 34% organic carbon converted into CO2 gas during four-hour oxidation. 2,4-Dichlorophenol (2,4-DCP), phenol, benzyl alcohol and two unknowns (RT = 2.65 and 3.78 min.) were detected as aromatic intermediates while Phenol was the new aromatic in HPLC analysis. Dechlorination efficiencies were high (> 70%) in all the cases, and more than 90% efficiencies were observed in chloride mass balance. Bigger flow rates and solution temperature fixed at 20 degrees C without pH adjustment greatly enhanced 2,4-D mineralization. These results can be an important basis in applying the treatment method for dioxin-contaminated water and wastewater.
Subject(s)
2,4-Dichlorophenoxyacetic Acid/chemistry , Herbicides/chemistry , Titanium/chemistry , Ultraviolet Rays , Water Pollutants, Chemical/chemistry , Water Purification/methods , 2,4-Dichlorophenoxyacetic Acid/metabolism , Catalysis , Herbicides/metabolism , Heterocyclic Compounds, 1-Ring/chemistry , Hydrogen-Ion Concentration , Oxidation-Reduction , Photochemistry , Waste Disposal, Fluid , Water Pollutants, Chemical/metabolism , Water Purification/instrumentationABSTRACT
Alpinia zerumbet (K. Schum), a medicinal plant originated from West Asia, is used in the northeast and southeast of Brazil as infusions or decoctions as a diuretic, antihypertensive, and antiulcerogenic. Experiments were undertaken to determine whether a hydroalcoholic extract obtained from leaves of Alpinia zerumbet (AZE) induces vasodilation in the mesenteric vascular bed (MVB), and an antihypertensive effect was also assessed in rats with DOCA-salt hypertension. In MVB precontracted with norepinephrine, AZE induces a long-lasting endothelium-dependent vasodilation that is not reduced by indomethacin. Inhibition of NO synthase by NG-nitro-L-arginine methyl ester (L-NAME) and guanylyl cyclase by 1H-[1,2,3]oxadiazolo [4,4-a]quinoxalin-1-one (ODQ) reduces the vasodilator effect of AZE. In vessels precontracted with norepinephrine, the vasodilator effect of AZE was not changed by 4-aminopyridine, glibenclamide, or by charybdotoxin plus apamin. Concentrations of atropine, pyrilamine, and yohimbine that significantly reduced the vasodilator effect of acetylcholine, histamine, and clonidine, respectively, did not change the vasodilator effect of AZE. HOE 140, which significantly reduced the vasodilator effect of bradykinin, induced a slight but significant reduction on the vasodilator effect of AZE. Chronic oral administration of AZE induced a significant reduction in systolic, mean, and diastolic arterial pressure in rats with DOCA-salt hypertension. Probably the vasodilator effect of AZE is dependent on the activation of the NO-cGMP pathway and independent of activation of ATP-dependent, voltage-dependent, and calcium-dependent K+ channels. Bradykinin receptors may also participate in the vasodilator effect of AZE. Finally, the vasodilator and antihypertensive effects of AZE demonstrated in the present study provide experimental support for the indication of Alpinia zerumbet as an antihypertensive medicinal plant.
Subject(s)
Alpinia/chemistry , Antihypertensive Agents/pharmacology , Endothelium, Vascular/physiology , Vasodilator Agents/pharmacology , Acetylcholine/pharmacology , Animals , Blood Pressure/drug effects , Desoxycorticosterone , Dose-Response Relationship, Drug , Drug Interactions , Hypertension/chemically induced , Hypertension/drug therapy , Hypertension/physiopathology , In Vitro Techniques , Male , Mesenteric Arteries/drug effects , Plant Extracts/pharmacology , RatsABSTRACT
1. The mechanisms involved in the vasodilator actions of angiotensin II (Ang II) have not yet been completely elucidated. We investigated the potential mechanisms that seem to be involved in the Ang II vasodilator effect using rat isolated mesenteric vascular bed (MVB). 2. Under basal conditions, Ang II does not affect the perfusion pressure of MVB. However, in vessels precontracted with norepinephrine, Ang II induces vasodilation followed by vasoconstriction. Vasoconstrictor, but not the vasodilation of Ang II, is inhibited by AT(1) antagonist (losartan). The vasodilator effect of Ang II was not inhibited by AT(2), angiotensin IV and angiotensin 1-7 receptor antagonists alone (PD 123319, divalinal, A 779, respectively). 3. The vasodilator effect of Ang II is significantly reduced by endothelial removal (deoxycholic acid), but not by indomethacin. Inhibition of NO-synthase by N(G)-nitro-l-arginine methyl ester (l-NAME) and guanylyl cyclase by 1H-[1,2,3] oxadiazolo [4,4-a] quinoxalin-1-one (ODQ) reduces the vasodilator effect of Ang II. This effect is also reduced by tetraethylammonium (TEA) or l-NAME, and a combination of l-NAME plus TEA increases the inhibitory effect of the antagonists alone. However, indomethacin does not change the residual vasodilator effect observed in vessels pretreated with l-NAME plus TEA. 4. In vessels precontracted with norepinephrine and depolarized with KCl 25 mm or treated with Ca(2+)-dependent K(+) channel blockers (charybdotoxin plus apamin), the effect of Ang II was significantly reduced. However, this effect is not affected by ATP and voltage-dependent K(+) channel blockers (glybenclamide and 4-aminopyridine). 5. Inhibition of kininase II with captopril significantly potentiates the vasodilator effect of bradykinin (BK) and Ang II in the rat MVB. The inhibitory effect of the B(2) receptor antagonist HOE 140 on the vasodilator effect of Ang II is further enhanced by PD 123319 and/or A 779. 6. The present findings suggest that BK plays an important role in the endothelium-dependent vasodilator effect of Ang II. Probably, the link between Ang II and BK release is modulated by receptors that bind PD 123319 and A 779.
Subject(s)
Angiotensin II/pharmacology , Angiotensin I/physiology , Bradykinin/physiology , Endothelium-Dependent Relaxing Factors/metabolism , Peptide Fragments/physiology , Receptor, Angiotensin, Type 2/physiology , Vasodilation/physiology , Animals , In Vitro Techniques , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiology , Mesenteric Veins/drug effects , Mesenteric Veins/physiology , Rats , Rats, Wistar , Receptors, Angiotensin/physiology , Vasodilation/drug effectsABSTRACT
Cumulative evidence suggests that moderate wine consumption exerts a cardioprotective effect. We investigated the occurrence of an antihypertensive effect of an alcohol-free hydroalcoholic grape skin extract (GSE) obtained from skins of a vinifera grape (Vitis labrusca) in experimental rodent hypertension models. The vasodilator effect of GSE (polyphenols concentration 55.5 mg g(-1)) was also assessed in the isolated mesenteric vascular bed of Wistar rats and the antioxidant effect was studied on lipid peroxidation of hepatic microsomes. Oral administration of GSE significantly reduced systolic, mean and diastolic arterial pressure in Wistar rats with desoxycorticosterone acetate-salt and N(G)-nitro-L-arginine methyl ester (L-NAME) induced experimental hypertension. In the rat isolated mesenteric vascular bed pre-contracted with norepinephrine, bolus injections of GSE induced endothelium-dependent vasodilatation that was substantially inhibited by L-NAME, but not by indometacin, tetraethylammonium or glibenclamide. Lipid peroxidation of hepatic microsomes estimated as malondialdehyde production was concentration-dependently inhibited by GSE. In conclusion, the antihypertensive effect of GSE might be owing to a combination of vasodilator and antioxidant actions of GSE. These findings also suggest that the beneficial effect of moderate red wine consumption could be owing to an antihypertensive action induced by compounds occurring in the skin of vinifera grapes.
Subject(s)
Antihypertensive Agents/therapeutic use , Antioxidants/therapeutic use , Flavonoids , Hypertension/drug therapy , Phytotherapy , Plant Extracts/therapeutic use , Vasodilator Agents/therapeutic use , Vitis/chemistry , Animals , Antihypertensive Agents/pharmacology , Antioxidants/pharmacology , Hypertension/chemically induced , Hypertension/physiopathology , In Vitro Techniques , Lipid Peroxidation/drug effects , Male , Mesenteric Artery, Superior/drug effects , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Phenols/analysis , Plant Extracts/chemistry , Polymers/analysis , Polyphenols , Rats , Rats, Wistar , Time Factors , Vasodilation/drug effects , Vasodilator Agents/pharmacology , Water , WineABSTRACT
We isolated the lipoteichoic-acid-related molecule (OK-PSA) from OK-432, a streptococcal preparation, by affinity chromatography on CNBr-activated Sepharose-4B-bound monoclonal antibody TS-2, which neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. We have previously reported that OK-PSA is a potent inducer of Th1-type cytokines in human peripheral blood mononuclear cells in vitro. In this study, we conducted an animal experiment to examine whether OK-PSA exhibits an anti-tumor effect in vivo by acting as a Th1 inducer in syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. It was found that OK-PSA induced Th1-type cytokines [IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-2, IL-12 and IL-18] in BALB/c mice bearing Meth-A tumor and caused a marked anti-tumor effect. Although it was suggested by an in vitro study. using spleen cells derived from the animals, that IL-18 plays the greatest role in the induction of the Th1-dominant state and tumor cell killing induced by OK-PSA, the in vivo experiments demonstrated that both IL-12 and IL-18 are essential in the anti-tumor effect exhibited by OK-PSA. These findings strongly suggest that OK-PSA is a major effector molecule of OK-432 and may be a useful immunotherapeutic agent, as a potent Th1 inducer, for cancer patients with a Th2-dominant state.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Antineoplastic Agents/therapeutic use , Lipopolysaccharides/therapeutic use , Streptococcus pyogenes/immunology , Teichoic Acids/therapeutic use , Th1 Cells/drug effects , Adjuvants, Immunologic/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/isolation & purification , Apoptosis , Chromatography, Affinity , Drug Screening Assays, Antitumor , Fas Ligand Protein , Female , Fibrosarcoma/chemically induced , Fibrosarcoma/immunology , Fibrosarcoma/pathology , Fibrosarcoma/therapy , Interleukin-12/antagonists & inhibitors , Interleukin-12/physiology , Interleukin-18/antagonists & inhibitors , Interleukin-18/physiology , Killer Cells, Natural/immunology , Lipopolysaccharides/chemistry , Lipopolysaccharides/pharmacology , Lymphokines/blood , Lymphokines/metabolism , Lymphoma/immunology , Lymphoma/pathology , Lymphoma/therapy , Lymphoma/virology , Membrane Glycoproteins/physiology , Mice , Mice, Inbred BALB C , Moloney murine leukemia virus , Neoplasm Transplantation , Penicillin G/pharmacology , Perforin , Picibanil/chemistry , Pore Forming Cytotoxic Proteins , Spleen/immunology , Spleen/pathology , Streptococcus pyogenes/chemistry , Streptococcus pyogenes/drug effects , Teichoic Acids/chemistry , Teichoic Acids/pharmacology , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/metabolism , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolism , fas Receptor/physiologyABSTRACT
We previously generated a monoclonal antibody, TS-2, that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432, a penicillin-killed streptococcal preparation [J. Immunother. 13 (1993) 232]. Expression of the TS-2-binding antigen was markedly higher in the cell wall of the penicillin-treated Streptococcus pyogenes (OK-432) than in the untreated bacteria (Su-BBM). We here isolated the antigens from OK-432 and Su-BBM, designated OK-PSA and Su-PSA, respectively. OK-432 markedly induced IFN-gamma and interleukin (IL)-18 as compared with Su-BBM in human peripheral blood mononuclear cells (PBMC). Furthermore, all of the Thl-type and Th1-inducing cytokines tested [IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-12 and IL-18] were secreted by OK-PSA-stimulated PBMC far better than by Su-PSA-treated PBMC. In addition, the cytolytic activities of the PBMC were accelerated by the stimulation with OK-432 or OK-PSA far better than by the stimulation with Su-BBM or Su-PSA. These findings strongly suggested that OK-PSA is a highly important molecule of OK-432 and may be a useful immunotherapeutic agent for the patients with malignant diseases as a potent Th inducer. It was also shown that penicillin treatment effectively enhances OK-PSA-induced anti-cancer immunity.
Subject(s)
Cytokines/biosynthesis , Lipopolysaccharides/metabolism , Penicillins/pharmacology , Picibanil/pharmacology , Streptococcus pyogenes/chemistry , Streptococcus pyogenes/metabolism , Teichoic Acids/metabolism , Caspase 1/metabolism , Cell Survival/drug effects , Culture Media , DNA Primers/pharmacology , Humans , In Vitro Techniques , Microscopy, Immunoelectron , Monocytes/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Th1 Cells/drug effects , Th1 Cells/metabolismABSTRACT
UNLABELLED: The mechanism underlying the Niteroi, Rio de Janeiro sedative effect of clonidine, an alpha2-adrenoceptor agonist, remains uncertain. Because activation of alpha2-adrenoceptors induces release of nitric oxide (NO), we tested the hypothesis that the sedative effect of clonidine depends on NO-related mechanisms. The effect of 7-nitro indazole on the sleeping time induced by clonidine was studied in Wistar rats. In addition, we examined the effect of clonidine, alpha-methyldopa, and midazolam on the thiopental-induced sleeping time in rats pretreated with N(G)-nitro-L-arginine-methyl-ester (L-NAME). The sleeping time induced by clonidine was significantly decreased by 7-nitro indazole. Thiopental sleeping time was increased by clonidine, alpha-methyldopa, and midazolam. L-NAME reduced the prolongation effect of clonidine and alpha-methyldopa, but did not alter the effect of midazolam on the thiopental-induced sleeping time. The inhibitory effect of L-NAME on clonidine-dependent prolongation of thiopental-induced sleeping time was reversed by L-arginine. These results suggest that NO-dependent mechanisms are involved in the sedative effect of clonidine. In addition, this effect seems to be specific for the sedative action of alpha2-adrenoceptors agonists. IMPLICATIONS: Clonidine, an antihypertensive drug, is also a sedative. This sedative effect, although an adverse event in the treatment of hypertensive patients, can be helpful for sedation of surgical patients. The mechanism of this effect, however, is unknown. In this study, we show that the sedative effect of clonidine is mediated by nitric oxide, because it could be prevented by pretreatment with nitric oxide synthase inhibitors.
Subject(s)
Adrenergic alpha-Agonists/pharmacology , Clonidine/pharmacology , Enzyme Inhibitors/pharmacology , Indazoles/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Animals , Dose-Response Relationship, Drug , Drug Interactions , Hypnotics and Sedatives/pharmacology , Male , Methyldopa/pharmacology , Midazolam/pharmacology , Nitric Oxide/physiology , Nitric Oxide Synthase Type I , Rats , Sleep/drug effects , Thiopental/pharmacologyABSTRACT
A lipoteichoic acid-related molecule (OK-PSA) isolated from OK-432, a penicillin-killed Streptococcus pyogenes, is a potent inducer of Th1 cytokines, and elicits anti-cancer effect in tumor-bearing mice. Toll-like receptor (TLR) 4 is a member of the recently identified toll-like receptor family of proteins that has been implicated in lipopolysaccharide-induced cell signaling. In the present study, we have examined the role of TLR4 for OK-PSA-induced Th1-cytokine production and anti-tumor effect by using C3H/HeJ mice in which TLR4 function is impaired. Although OK-PSA strikingly induced Th1 cytokines [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18] in the splenocytes derived from control animals (C3H/HeN), OK-PSA did not induce the cytokines in the splenocytes from C3H/HeJ. Furthermore, C3H/HeJ-derived splenocytes acquired the responsiveness to OK-PSA stimulation by overexpression of TLR4 gene. Finally, OK-PSA administration significantly inhibited the tumor growth and lung metastasis of syngeneic squamous cell carcinoma cells in C3H/HeN; however, no effect of OK-PSA was observed in C3H/HeJ. These findings strongly suggest that TLR4 signaling is involved in regulating OK-PSA-induced anti-cancer immunity.
Subject(s)
Antineoplastic Agents/pharmacology , Drosophila Proteins , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/deficiency , Penicillins/pharmacology , Picibanil/chemistry , Receptors, Cell Surface/deficiency , Streptococcus pyogenes/chemistry , Teichoic Acids/pharmacology , Animals , Antineoplastic Agents/chemistry , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/pathology , Cell Line , Culture Media , Lipopolysaccharides/chemistry , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C3H , Mice, Knockout , Mutation/genetics , Receptors, Cell Surface/genetics , Spleen/cytology , Spleen/drug effects , Spleen/immunology , Streptococcus pyogenes/drug effects , Teichoic Acids/chemistry , Toll-Like Receptor 4 , Toll-Like Receptors , Transfection , Tumor Cells, CulturedABSTRACT
We have isolated a 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by affinity chromatography on an N-hydroxysuccinimide-activated Sepharose High Performance column bound with F3, a monoclonal antibody that neutralizes the cytokine-inducing and anti-tumor effect of AIL. In the present study, we examined this protein (AILb-A) for cytokine induction and anti-tumor effects by animal study, using syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. AILb-A administration resulted in markedly increased levels of Th1 cytokines [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18] in the sera derived from Meth-A-bearing mice. The in vitro re-stimulation with AILb-A of splenocytes derived from AILb-A-primed mice also selectively induced Th1-type cytokines and antigen-specific killer cell activity. The neutralizing test using cytokine-specific antibodies revealed that AILb-A-induced IL-18 plays a most significant role for and killer cell-inducing activities. Furthermore, IL-12 and IL-18 induced by AILb-A inhibited specifically IL-10 and IL-4 production, respectively. Finally, we examined the anti-tumor effect of AILb-A in both Meth-A-bearing BALB/c mice and Meth-A-bearing nude mice with BALB/c background. AILb-A exhibited a striking anti-tumor effect in normal BALB/c mice inoculated with Meth-A cells. In athymic nude mice, the anti-tumor effect of AILb-A was relatively weak. These findings strongly suggested that AILb-A is a potent Th1 inducer and may be a useful immunotherapeutic agent for patients with malignant diseases.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cytokines/biosynthesis , Neoplasms, Experimental/drug therapy , Th1 Cells/immunology , Animals , Antibodies/immunology , Antineoplastic Agents, Phytogenic/isolation & purification , Cells, Cultured , Cytokines/immunology , Cytotoxicity Tests, Immunologic , Female , Fibrosarcoma/immunology , Fibrosarcoma/therapy , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms, Experimental/immunology , Plant Proteins/isolation & purification , Plant Proteins/pharmacology , Spleen/immunology , Survival Rate , Th2 Cells/immunology , Tumor Cells, CulturedABSTRACT
3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K(m) values for 3-isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degrees C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.
ABSTRACT
A tetrathionate-decomposing enzyme that catalyzes the decomposition of tetrathionate into thiosulfate and sulfate was purified to homogeneity from tetrathionate-grown Thiobacillus thiooxidans. The enzyme had an apparent molecular weight of 104,000, and was composed of two identical subunits (MW = 58,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point at 9.6 and was most active at pH 3.0-3.5 and 40°C. Enzyme activity was increased approximately 100-fold in the presence of 400 mm sulfate ion. The Michaelis constant of this enzyme for tetrathionate in the presence of 20, 50, and 200 mm of sulfate ion was 2.4 mm. Mercuric and ferric ions completely inhibited the enzyme activity at 1 mm. Though cupric ion up to 0.01 mm markedly stimulated the activity in the presence of 20 mm sulfate ion, a higher concentration (1 mm) rather strongly inhibited the activity. Ethylenediaminetetraacetic acid (EDTA) strongly inhibited the activity, but this inhibiton was completely restored by cupric ion.
ABSTRACT
The phylogenetic position of an acidophilic chemo-organotrophic menaquinone-containing bacterium, Acidobacterium capsulatum, was studied on the basis of 16S rRNA gene sequence information. A. capsulatum showed the highest level of sequence similarity to Heliobacterium chlorum, a member of the Gram-positive group, yet this level was only 81%. Distance matrix tree analysis suggested that A. capsulatum belongs to a unique lineage deeply branching from the Chlamydia-Planctomyces group or from the Gram-positive line.
Subject(s)
Evolution, Molecular , Gram-Negative Chemolithotrophic Bacteria/genetics , Phylogeny , Vitamin K , Base Sequence , Gram-Negative Chemolithotrophic Bacteria/metabolism , Molecular Sequence Data , RNA, Bacterial/chemistry , RNA, Ribosomal, 16S/chemistry , Sequence Analysis, RNA , Vitamin K/biosynthesisABSTRACT
Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space. The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T. ferrooxidans AP19-3. When pTHS1 was used to transform sulfite reductase deficient E. coli mutants, strain AT2455 (cysG), JM246 (cysI), and AT2427 (cysJ), it complemented only the E. coli cysG mutation. Since cysG codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase is present in a T. ferrooxidans 1.3 kb DNA fragment on pTHS1.
Subject(s)
Escherichia coli/genetics , Genetic Complementation Test , Methyltransferases/genetics , Thiobacillus/genetics , DNA, Bacterial , Mutation , NAD/metabolism , Oxidoreductases Acting on Sulfur Group Donors/metabolism , PlasmidsABSTRACT
When grown on iron-salt medium supplemented with the bisulfite ion, Leptospirillum ferrooxidans was much more sensitive to the ion than was Thiobacillus ferrooxidans. The causes of the sensitivity of L. ferrooxidans to the bisulfite ion were studied. The bisulfite ion completely inhibited the iron-oxidizing activities of L. ferrooxidans and T. ferrooxidans at 0.02 and 0.2 mM, respectively. A trapping reagent for the bisulfite ion, formaldehyde, completely reversed the inhibition. The treatment of intact cells with 1.0 mM bisulfite ion for 1 h and washing the bisulfite ion from the cells had no harmful effects on the iron-oxidizing activity of T. ferrooxidans. However, the treatment of L. ferrooxidans with 0.1 mM bisulfite ion for 1 h completely destroyed the iron-oxidizing activity. T. ferrooxidans had sulfite:ferric ion oxidoreductase activity. In contrast, a quite low level of sulfite:ferric ion oxidoreductase activity was found in L. ferrooxidans, suggesting that it is much more difficult for L. ferrooxidans to oxidize the bisulfite ion to the less harmful sulfate than it is for T. ferrooxidans. These results suggest that the sensitivity of L. ferrooxidans to the bisulfite ion is due to a lack of an active sulfite:ferric ion oxidoreductase and the sensitivity of its iron oxidase to bisulfite ion.
ABSTRACT
A restriction endonuclease, Aor13HI, an isoschizomer of BspMII, was purified to homogeneity from cell extracts of Acidiphilium organovorum strain 13H. The enzyme has a molecular mass of 60,000 daltons and consists of two subunits identical in molecular mass of 30,000 daltons. Aor13HI endonuclease, like BspMII, recognizes the palindromic six-base sequence 5'-TCCGGA-3', and cleaves between the T and C to produce a four-base 5' extension. Aor13HI is not inhibited by dam-dependent methylation. The isoelectric point of the enzyme is 5.7. Aor13HI activity was maximum at pH 7.5, 100 mM KCl, 7.5-10 mM MgCl2, and 55 degrees C. The enzyme was stable up to 60 degrees C. The N-terminal amino acid sequence (30 residues) of Aor13HI did not show any similarity with the sequence of other restriction endonucleases reported.