Radioprotective and antioxidant properties of low-altitude Podophyllum hexandrum (LAPH).

The development of nontoxic yet effective radioprotectors is needed because of the increasing risk of human exposure to ionizing radiation. We have reported that high-altitude Podophyllum hexandrum (HAPH) confers a radioprotective effect in in vitro and in vivo models. The present study reports on the antioxidant and radioprotective properties of low-altitude Podophyllum hexandrum (LAPH), from which the toxic compound podophyllotoxin has been partially removed during the extraction process. Using HPLC,we estimated the relative content of two marker compounds, podophyllotoxin and podophyllotoxin glycoside, in low-altitude Podophyllum extract (LAPE) and found them to be 23.3% and 9.50%, respectively. The ferrous ion chelation potential of LAPE was estimated using the 2,2 bipyridyl assay, and the activity was found to be increased concomitantly with the increase in its concentration, with a maximal inhibition at 25 microg/mL (42.20%) as compared to quercetin (34.9%). The electron donation potential of LAPE was also evaluated, because the antioxidant activities of natural products are known to bear a direct correlation with their ability to donate electrons. The concentration required to attain unit absorbance values at 700 nm were 0.230541+/-0.09 and 0.041+/-0.06 for butylated hydroxyl toluene and LAPE, respectively, indicating a higher antioxidant activity of LAPE. The free radical scavenging ability of LAPE was also assessed and exhibited a dose-dependant increase (1-100 microg/mL), comparable to that of quercetin at 25 microg/mL. The role of LAPE in protecting DNA was evaluated, and it was found that LAPE (30 microg/mL) rendered its maximum radioprotection against the 250 Gy-induced damage in the plasmid (pBR322) relaxation assay. LAPE significantly inhibited radiation-induced, iron/ascorbate- and combined stress (iron/ascorbate and radiation)-induced formation of TBARS (p<0.05). We conclude that LAPH, with its easy accessibility, ease of cultivation, multifarious radioprotective properties, and role as a renewable source of bioactive constituents, along with its low associated toxicity (due to partial removal of podophyllotoxin), enhances its possible use for human clinical applications.     

Evaluation of the radioprotective effect of Aegle marmelos (L.) Correa in cultured human peripheral blood lymphocytes exposed to different doses of gamma-radiation: a micronucleus study

The radioprotective effect of a hydroalcoholic extract of Aegle marmelos (AME) was evaluated in cultured human peripheral blood lymphocytes (HPBLs) by the micronucleus assay. The optimum protective dose of the extract was selected by treating HPBLs with 1.25, 2.5, 5, 6.25, 10, 20, 40, 60, 80 and 100 microg/ml AME before exposure to 3 Gy gamma-radiation and then evaluating the micronucleus frequency in cytokinesis blocked HPBLs. Treatment of HPBLs with different doses of AME reduced the frequency of radiation-induced micronuclei significantly, with the greatest reduction in micronucleus induction being observed for 5 microg/ml AME. Therefore, this dose of AME was considered as the optimum dose for radioprotection and further studies were carried out treating the HPBLs with 5 microg/ml AME before exposure to different doses (0, 0.5, 1, 2, 3 and 4 Gy) of gamma-radiation. The irradiation of HPBLs with different doses of gamma-radiation caused a dose-dependent increase in the frequency of lymphocytes bearing one, two and multiple micronuclei, while treatment of HPBLs with 5 microg/ml AME significantly reduced the frequency of lymphocytes bearing one, two and multiple micronuclei when compared with the irradiated control. The dose-response relationship for both groups was linear. To understand the mechanism of action of AME separate experiments were conducted to evaluate the free radical scavenging of OH, O2(-), DPPH, ABTS(+) and NO in vitro. AME was found to inhibit free radicals in a dose-dependent manner up to a dose of 200 microg/ml for the majority of radicals and plateaued thereafter. Our study demonstrates that AME at 5 microg/ml protected HPBLs against radiation-induced DNA damage and genomic instability and its radioprotective activity may be by scavenging of radiation-induced free radicals and increased oxidant status.     

Effect of mangiferin on radiation-induced micronucleus formation in cultured human peripheral blood lymphocytes

Irradiation causes a variety of lesions in important biomolecules of the cell through generation of free radicals leading to genomic instability. DNA strand breaks, acentric fragments, or defective kinetochores are manifested as micronuclei after the first cell division. Chemicals that can trap free radicals may reduce the deleterious effects of ionizing radiation. Mangiferin (MGN), a glucosylxanthone derived from Mangifera indica (mango), was investigated for its ability to reduce the frequency of radiation-induced micronucleated binucleate cells (MNBNCs) in cultured human peripheral blood lymphocytes (HPBLs). HPBL cultures were pretreated with 0, 5, 10, 20, 50, and 100 microg/ml of MGN for 30 min before exposure to 3 Gy of (60)Co gamma-radiation. The maximum decline in radiation-induced micronuclei was observed at a concentration of 50 microg/ml MGN; thereafter, a nonsignificant elevation in MNBNC frequency was observed at 100 microg/ml MGN. Since the lowest MNBNC frequency was observed for 50 microg/ml MGN, dose-response studies were undertaken using this concentration. Irradiation of HPBLs with 0, 1, 2, 3, or 4 Gy of gamma-radiation caused a dose-dependent elevation in the MNBNC frequency, while treatment of HPBLs with 50 microg/ml MGN 30 min before radiation resulted in significant declines in these frequencies. MGN alone did not alter the proliferation index. Irradiation caused a dose-dependent decline in the proliferation index, while treatment of HPBLs with 50 micro/ml MGN significantly elevated the proliferation index in irradiated cells. MGN treatment reduced hydrogen peroxide-induced lipid peroxidation in HPBLs in a concentration-dependent fashion. In cell-free studies, MGN inhibited the induction of (.)OH (hydroxyl), O(2) (.-) (superoxide), DPPH (1,1-diphenyl-2-picrylhydrazyl), and ABTS(.+) (2,2-azino-bis-3-ethyl benzothiazoline-6-sulphonic acid) radicals in a dose-dependent manner. The results of this study indicate that MGN possesses radioprotective properties by suppressing the effects of free radicals.     

Radioprotective effects of thiomethylhydantoin derivatives on Escherichia coli and mice

Protection of Escherichia coli NIHJ and C57BL mice from the effects of 60Co gamma-rays provided by S-alk(en)yl-L-cysteines and their hydantoin derivatives was examined. E. coli (10(6) cells/ml) suspended in a 20 mM aqueous solution of one of the drugs was irradiated with 60 Gy of gamma-rays. Five week-old male mice were exposed to 5.0-9.5 Gy of gamma-rays after a single intraperitoneal injection of 0.75 mmol/kg body weight of each compound. In both E. coli and mice, S-allyl compounds afforded more effective radioprotection than S-propyl compounds. The replacement of the alpha-hydrogen of S-substituted cysteines by methyl groups decreased the radioprotective effect. Hydantoin derivatives were much more radioprotective than the original sulfur-containing amino acids. Especially, DL-5-allylthiomethyl-5-methylhydantoin had a remarkable radioprotective effect in mice. The gamma-radiolysis mechanism of thiomethylhydantoin derivatives was discussed in connection with the radioprotective effect of the drugs.     

Induction of apoptosis in thymocytes by Hippophae rhamnoides: implications in radioprotection.

Hippophae rhamnoides (RH-3), which has been recently reported to elicit dose-dependent pro- and antioxidant properties in vitro, induced apoptosis in murine thymocytes. In a concentration-dependent manner, RH-3 induced apoptosis in thymocytes in ex vivo conditions. The maximum effect was observed with 100 microg/mL of RH-3. Beyond this dose, the induction of apoptosis was inhibited, as seen on the ladder formation. However, apoptotic body formation, another indicator of apoptosis, was not manifested when various doses of RH-3 (20-200 microg/mL) were administered. RH-3 (>100 microg/mL) compacted chromatin in the form of densely stained masses, and subsequent treatment with proteinase-K loosened them and developed a halo around each mass. RH-3 treatment of cells that had already undergone apoptosis induced chromatin compaction, which made the ladder invisible. During in vivo experiments in mice, the radioprotective dose of RH-3 (30 mg/kg b.w.) induced significant DNA fragmentation in thymocytes studied spectrofluorimetrically. RH-3 treatment before irradiation in vivo enhanced radiation-induced apoptosis. These results were confirmed by hypodiploid population studied flow-cytometrically and also by ladder formation. RH-3 treatment was prooxidative in nature because it depleted thiols and enhanced lipids peroxidation after 8 hours of treatment. The paradox between the prooxidant and the antioxidant effects of RH-3 in the context of its overall radioprotective efficacy has been explained.     

Protection of ionizing radiation-induced cytogenetic damage by hydroalcoholic extract of Cynodon dactylon in Chinese hamster lung fibroblast cells and human peripheral blood lymphocytes.

The radiomodulatory potential of hydroalcoholic extract of a medicinal plant Cynodon dactylon (family: Poaceae) against radiation-induced cytogenetic damage was analyzed using Chinese hamster lung fibroblast (V79) cells and human peripheral blood lymphocytes (HPBLs) growing in vitro. Induction of micronuclei was used as an index of cytogenetic damage, evaluated in cytokinesis blocked binucleate cells. The hydroalcoholic Cynodon dactylon extract (CDE) rendered protection against the radiation-induced DNA damage, as evidenced by the significant (p<0.001) reduction in micronucleated binucleate cells (MNBNC%) after various doses of CDE treatment in V79 cells and HPBLs. The optimum dose of CDE (40 and 50 microg/ml in HPBLs and V79 cells, respectively) with the greatest reduction in micronuclei was further used in combination with various doses of gamma radiation (0.5, 1, 2, 3, and 4 Gy) exposed 1 h after CDE treatment. A linear dose-dependent MNBNC% increase in radiation alone group was observed, while 40/50 microg/ml CDE significantly resulted in the reduction of MNBNC%, compared to the respective radiation alone groups. CDE resulted in a dose-dependent increase in free radical scavenging ability against various free radicals, viz., 2, 2-diphenyl-2-picryl-hydrazyl (DPPH); 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); superoxide anion (O2*-); hydroxyl radical (OH*) and nitric oxide radical (NO*) generated in vitro. Also, an excellent (70%) inhibition of lipid peroxidation in vitro was observed at a dose of 300 microg/ml CDE, attaining the saturation point at higher doses. The present findings demonstrated the radioprotective effect of CDE, also rendering protection against radiation-induced genomic instability and DNA damage. The observed radioprotective effect may be partly attributed to the free radical scavenging and antilipid peroxidative potential of CDE.     

Antioxidant, anticlastogenic and radioprotective effect of Coleus aromaticus on Chinese hamster fibroblast cells (V79) exposed to gamma radiation

Coleus aromaticus (Benth, Family: Laminaceae), Indian Oregano native to India and Mediterranean, is well known for its medicinal properties. A preliminary study was undertaken to elucidate in vitro free radical scavenging potential and inhibition of lipid peroxidation by C.aromaticus hydroalcoholic extract (CAE). Anti-clastogenic and radioprotective potential of CAE were studied using micronucleus assay after irradiating Chinese hamster fibroblast (V79) cells. CAE at 10, 20, 40, 60, 80, 100 and 120 mug/ml resulted in a dose-dependent increase in radical scavenging ability against various free radicals viz., 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), superoxide anion (O(2)(*-)), hydroxyl (OH(*)) and nitric oxide (NO(*)) generated in vitro. A maximum scavenging potential was noticed at 100 mug/ml and a saturation point was reached thereafter with the increasing doses of CAE. The free radical scavenging potential of the extract was in the order of DPPH > ABTS > Superoxide > Hydroxyl > Nitric oxide. CAE also exhibited a moderate inhibition of lipid peroxidation in vitro, with a maximum inhibition at 60 mug/ml (33%), attaining saturation at higher doses. The extract also rendered protection against radiation induced DNA damage, as evidenced by the significant (P < 0.05) decrease in the percentage of radiation-induced micronucleated cells (MN) and frequency of micronuclei (total). A maximum anticlastogneic effect/ radioprotection was noticed at a very low concentration i.e., 5 mug/ml of CAE, treated 1 h prior to 2 Gy of gamma radiation. A significant (P < 0.0001) anticlastogenic/radioprotective effect was also observed when the cells were treated with an optimum dose of CAE (5 mug/ml) 1 h prior to 0.5, 1, 2 and 4 Gy of gamma radiation compared with the respective radiation control groups. Overall, our results established an efficient antioxidant, anticlastogenic and radioprotective potential of CAE, which may be of great pharmacological importance.     

Vangueria infausta root bark: in vivo and in vitro antiplasmodial activity

Vangueria infausta burch subsp. infausta (Rubiaceae) produces fruits eaten by humans and animals. The leaf, fruit, stem bark and root bark are used as a remedy for many ailments and the roots are used to treat malaria. In this study, concentrations of fractions of the V. infausta root bark extract that produce 50% inhibition (IC50) are determined using the ability of the extract to inhibit the uptake of [G3H]-hypoxanthine by P. falciparum cultured in vitro. The root bark extract showed antimalarial activity against Plasmodium berghei in mice. It gave a parasite suppression of 73.5% in early infection and a repository effect of 88.7%. One fraction obtained from a chloroform extract gave an IC50 value of 3.8 +/- 1.5 microg/mL and 4.5 +/- 2.3 microg/mL against D6 and W2 strains of P. falciparum, respectively, and another from the butanol extract gave an IC50 value of 3.9 +/- 0.3 microg/mL against the D6 strain. Chloroquine had an IC50 value of 0.016 microg/mL and 0.029 microg/mL against D6 and W2 strains, respectively. The plant showed the presence of flavonoids, coumarins, tannins, terpenoids, anthraquinones and saponins.     

Unique radiosensitivity of serous cells in rhesus monkey submandibular glands.

The saliva of patients undergoing radiation therapy for head and neck cancer contains increased acidic mucosubstances associated with a reduced serous component. To assess the morphologic features of the acute radiation damage in serous versus mucous acinar cells, the mixed serous/mucous submandibular glands of 18 rhesus monkeys were studied 1-72 hours after irradiation with single doses of 2.5-15.0 Gy. Selective degeneration and necrosis of serous cells was observed with doses of 2.5-7.5 Gy. Doses of 10.0-15.0 Gy caused widespread destruction of whole serous acini, but only isolated mucous cells were affected. The lesions were clearly expressed by 24 hours. Transient exudation of neutrophils was replaced by plasma cells and lymphocytes. Examination at 16, 22 and at 40 weeks revealed that late atrophy was caused solely by loss of serous acini in glands treated with 7.5 and 10.0 Gy. Although both serous and mucous acini were reduced in glands treated with 12.5 and 15.0 Gy, the atrophy was mainly due to loss of serous acini. The finding that serous cells are more vulnerable to radiation injury than mucous cells provides a morphologic explanation for early and late changes in saliva composition after salivary gland irradiation.     

Acute and late radiation injury in rhesus monkey parotid glands. Evidence of interphase cell death.

Acute and chronic salivary gland dysfunction are common sequelae of radiotherapy for head and neck cancer; but the associated morphologic changes, especially of the acute damage, have received relatively little study. For investigation of the morphologic characteristics of acute radiation injury to parotid glands, rhesus monkeys were studied 1-72 hours after parotid irradiation with single doses of 2.5-15.0 Gy. The acute damage from all doses was clearly expressed by 24 hours. Histologically, parotid glands irradiated with 2.5 or 5.0 Gy had random degeneration and necrosis of the serous acinar cells. Doses of 7.5-15.0 Gy produced widespread degeneration along with necrosis of whole acini. Serous cell damage was accompanied by neutrophilic inflammation that subsided after 24 hours to become replaced by plasma cell and lymphocytic infiltrates. Parotid glands receiving 7.5-15.0 Gy were atrophic at 16-22 weeks after irradiation and showed no recovery by 40 weeks. Although parotid acinar cells are well-differentiated nondividing cells, these observations show that they express lethal radiation injury in interphase within hours of receiving a radiation dose as low as 2.5 Gy. This is unlike most mammalian cells that express radiation injury during mitosis. Chronic atrophy is a consequence of this direct, irreversible, and early injury, rather than the result of radiation-induced changes in the vasculature.     

The role of hematopoietic microenvironment in prodigiozan' mechanism of action on postradiation recovery of hemopoiesis in long-term bone marrow cultures

Influence of prodigiozan in different concentrations on the process of postirradiation recovery of haemopoietic precursors (GM-CFC) and morphologically recognizable elements of the bone marrow were studied in long-term bone marrow cultures during 28 days after 2 Gy gamma-irradiation. Also, prodigiozan was studied for action on the contents of GM-CFC and induction of GM-CSF three, 24 and 48 hours after injection in cultures unexposed to radiation. It is shown that injection of prodigiozan in concentration 0.1-1.0 microg/ml 24 hours prior to irradiation stimulates postradiation recovery of GM-CFC number and a total number of myelocaryocytes in irradiation of long-term bone marrow cultures. In saved cultures there was an increase in the number of stromal cells 1 and 2 days after radiation. In non-exposed to radiation long-term bone marrow cultures injection of prodigiozan increased induction of GM-CSF and raised contents of GM-CFC. The maximal increase occurred 24 hours after introduction of radioprotector. It is suggested that one of the mechanisms of a radioprotective action of prodigiozan may be stimulation of hematopoietic microenvironment cellular elements leading to a marked release of GM-CSF or/and other cytokines and stimulation of recovery of hemopoietic precursors.     

Accelerated recovery of hematopoiesis following sub-lethal whole body irradiation with recombinant murine interleukin-1 (IL-1)

This communication reports the results of studies designed to investigate the ability of recombinant murine interleukin-1 (rIL-1) to enhance the recovery of hematopoiesis following administration of sub-lethal whole body irradiation (2 Gy). Mice were administered rIL-1 (100 and 500 units) i.p. Twenty-four hours later these mice were administered 2 Gy radiation. Irradiated control mice were given only phosphate buffered saline (PBS). Animals were then serially sacrificed (on days 1, 2, 4, 7, 9, and 12 following irradiation) and their peripheral blood was analyzed for indices (packed red cell volume, WBC, platelets, and differential). Femoral bone marrow was harvested and assayed for their stem cell content--erythroid (CFU-E, BFU-E), granulocyte-macrophage (CFU-GM), and megakaryocyte (CFU-MEG). Irradiated mice pretreated with rIL-1 demonstrated accelerated hematopoietic recovery as measured by higher WBC, platelets and femoral stem cell content than PBS-treated irradiated controls. These results indicate IL-1 may be an effective radioprotective agent against the hematotoxicity induced by ionizing radiation.     

Cytotoxic effects against HeLa cells of polysaccharides from seaweeds

Polysaccharides extracted from several red and brown seaweeds growing along the South American coasts were evaluated regarding their cytotoxic effects against HeLa cells. Sulfated galactans were isolated and purified from the red algae Bostrychia montagnei (BHW and B4 fractions, 17 and 22% SO3Na, respectively) and Porphyra columbina (PC75 fraction, 15.6% SO3Na). At maximum concentration of 80 microg/mL, these fractions promoted atypical mitoses in HeLa cells, presence of atypical nuclei and blebs. Alginic acid (160.0 microg/mL) isolated from the brown seaweed Laminaria brasiliensis (molar ratio M/G 1.2) and its M and G blocks (40.0 microg/mL; DP = 20), promoted similar morphologic alterations besides the presence of acidophilic material in the cellular cytoplasm and occurrence of multinuclear cells present in the monolayer. The polysaccharide fraction SF isolated from the brown seaweed Sargassum stenophyllum containing mainly fucose and presenting molar ratio of sulfate/sugar 1.9, promoted very accentuated morphologic modifications in HeLa cells at low concentrations. SF (2.5 microg/mL) caused significant alterations in the cellular morphology and reduction of the growth, such effects being dose dependent. At 40.0 microg/mL, accentuated decrease of the number of mitosis illustrations accompanied by an increase in the number of condensed cells, atypical nuclei, number of clusters and blebs. These results demonstrated that among the anionic polysaccharides tested, the sulfated fucans were those which caused greater cytotoxic effects in HeLa cells.     

Prevention of Radiation Induced Hematological Alterations by Medicinal Plant Rosmarinus Officinalis, in Mice

The modulatory influence of Rosmarinus officinalis (rosemary) leaves extract was investigated in Swiss albino mice at a dose of 3 Gy gamma radiation. For this purpose, adult Swiss albino mice were irradiated with 3 Gy gamma rays in the presence (experimental) or absence (control) of rosemary (1000 mg/kg body wt.). These animals were necropsied and their blood was collected at days 1, 3, 5, 10, 20 and 30 post-irradiation. A decrease in the number of erythrocyte and leucocyte counts, hemoglobin content and hematocrit percentage was scored in the control group; whereas a recovery pattern was recorded in experimental animals and a normal value of hematological parameters were regained by day 30 post-treatment. In irradiated group, glutathione level was registered low in the blood, whereas a significant elevation was estimated in rosemary pre-treated animals. An increase in lipid peroxidation level above normal was evident in serum of irradiated mice, while a significant decrease in such values was noted in rosemary pretreated group. The present study suggests the possible radioprotective ability of rosemary extract.     

钙离子载体诱导HL-60细胞分化成树突状细胞的实验研究

目的 :探讨一种钙离子载体 (calciumionophore,CI)A2 3187,能否诱导早幼粒白血病细胞株HL 6 0分化成具有活性的树突状细胞 (dendriticcells,DCs)。方法 :将生长状态良好的HL 6 0细胞分别加在普通培养液中 ,或在含不同浓度 (2 5～ 16 0 0 μg/L)的A2 3187及 10 0 μg/L重组人粒 /单集落刺激因子 (rhGM CSF)的普通培养液中培养。 2 4～ 96h后 ,在光镜及电镜下观察细胞的形态 ;用流式细胞仪检测细胞的表面标志 ,MTT比色法检测其刺激同种异体T细胞增殖的作用。结果 :HL 6 0细胞以适量 (2 0 0 μg/L)的A2 3187诱导 2 4h ,DC的特征性表面标志CD83分子的表达最高 ,72h可出现典型的树突状突起 ,96h细胞表面CD80 (B7.1)、CD86 (B7.2 )、MHC II分子及细胞间黏附分子CD5 4的表达最高 ,且能明显激活同种异体T细胞。结论 :钙离子载体A2 3187可将HL 6 0细胞诱导成具有活性的DC样细胞。     

Safety, pharmacokinetics, and antiviral activity of AMD3100, a selective CXCR4 receptor inhibitor, in HIV-1 infection

AMD3100 is a CXCR4 receptor inhibitor with anti-HIV-1 activity in vitro. We tested the safety, pharmacokinetics, and antiviral effect of AMD3100 administered for 10 days by continuous intravenous infusion in an open-label dose escalation study from 2.5 to 160 microg/kg/h. Forty HIV-infected patients with an HIV RNA level >5000 copies/mL on stable antiretroviral (ARV) regimens or off therapy were enrolled. Syncytium-inducing (SI) phenotype in an MT-2 cell assay was required in higher dose cohorts. Most subjects were black (55%), male (98%), and off ARV therapy. HIV phenotype was SI (30%), non-SI (45%), or not tested (25%). One patient (5 microg/kg/h) had serious and possibly drug-related thrombocytopenia. Two patients (40 and 160 microg/kg/h) had unexpected, although not serious, premature ventricular contractions. Most patients in the 80- and 160-microg/kg/h cohorts had paresthesias. Steady-state blood concentration and area under the concentration-time curve were dose proportional across all dose levels; the median terminal elimination half-life was 8.6 hours (range: 8.1-11.1 hours). Leukocytosis was observed in all patients, with an estimated maximum effect of 3.4 times baseline (95% confidence interval: 2.9-3.9). Only 1 patient, the patient whose virus was confirmed to use purely CXCR4 and who also received the highest dose (160 microg/kg/h), had a significant 0.9-log10 copies/mL HIV RNA drop at day 11. Overall, however, the average change in viral load across all patients was +0.03 log10 HIV RNA. Given these results, AMD3100 is not being further developed for ARV therapy, but development continues for stem cell mobilization.     

Effect of hirudin versus heparin on hemocompatibility of blood contacting biomaterials: an in vitro study

Hirudin serves as an alternative anticoagulant for extracorporeal blood circulation. Comparing anticoagulation with hirudin (2.5 or 5.0 microg/mL) and heparin (2.0 or 4.0 IU/mL) human blood was circulated in a modified 'Chandler System' using PVC-tubes for 2 hours at 37 degrees C. Activation of coagulation (thrombin-antithrombin III-complex, prothrombin fragment 1+2 and D-Dimer), platelet (platelet factor 4 - PF4) and complement systems was analyzed. Both heparin concentrations and 5.0 microg/dL hirudin led to as significantly less activated plasmatic coagulation as 2.5 microg/dL hirudin. Decreased levels of PF4 and anaphylatoxin C5a (p<0.05) as well as terminal complement complex demonstrated improved hemocompatibility after anticoagulation with heparin in contrast to hirudin. Because initial coagulation cascade, platelet activation and complement activation is less influenced by hirudin than by heparin, hemocompatibility is more dependent on the characteristics of the biomaterials used. This predestines hirudin as anticoagulant for in vitro studies analyzing hemocompatibility of biomaterials or surface modifications.     

Hygromycin B-resistance phenotype acquired in Paracoccidioides brasiliensis via plasmid DNA integration.

Yeast cells of the human pathogenic fungus Paracoccidioides brasiliensis strain Pb01 were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved by electroporation, with intact or linearized plasmid DNA. The fungus was transformed using 200 mM manitol, 5 or 7 kV/cm field strength, 25 microF capacitance, 400 omega resistance, 5 microg plasmid DNA and 10(7) yeast cells in 400 microl, and selected in BHI medium overlaid with 30 microg/ml hygromycin B (hygB). Mitotic stability was assessed by growing transformants on non-selective BHI medium, followed by plating on hygromycin B (30 microg/ml). Transformants were analyzed by PCR and Southern blotting, confirming the hph gene integration into the transformants genome. A low level of stability of the integrated hph sequence in the transformant genomes was observed, probably because of the multinuclearity of P. brasiliensis yeast cells.     

The air pollutant sodium sulfite enhances mite crude extract-stimulated detachment of A549 airway epithelium cells.

BACKGROUND AND PURPOSE: It has been previously reported that the pollutant sodium sulfite (Na2SO3) can activate airway epithelial cells; however, there is as yet no evidence of any direct relationship between house dust mite allergen exposure and Na2SO3 with regards to the pathogenesis of airway allergy. This study investigated the effect of sulfite on mite-stimulated human airway epithelial cells. METHODS: The A549 human lung epithelial cell line was used as an in vitro model. Cells were treated with 10 microg/mL mite crude extract for 8 h and/or Na2SO3 (0, 10, 100, 500, 1000 and 5000 microM) for 16 h, and cell adhesion and dissociation on a cell culture plastic surface were quantitated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Changes in cell adhesion were also analyzed by monitoring the expression of the cell surface of adhesion molecules integrin alpha2 (CD49b) and integrin alpha6 (CD49f) using flow cytometry. RESULTS: A549 cells treated with either mite crude extract only or Na2SO3 only did not show a significant increase in dissociation from the cell culture plastic surface. However, when cells were pretreated with mite extract for 8 h, followed by 16-h incubation with various concentrations of Na2SO3, cell dissociation was enhanced in a dose-dependent manner. A dose-dependent decrease of CD49b and CD49f expression was also seen in cells treated with Na2SO3 only and in mite-pretreated cells. Mite treatment decreased CD49b expression, and a cumulative effect was seen in cells further treated with Na2SO3. CONCLUSION: Significant dissociation of airway epithelial cells with Na2SO3 stimulation only occurred in cells pretreated with mite extract. Mite pretreatment enhanced Na2SO3-induced CD49f down-regulation; Na2SO3 and/or mite extract down-regulated CD49b expression of A549 cells. These findings indicate that a synergistic effect of mite extract and sulfite can severely disrupt the airway bronchial epithelial barrier.     

Ultrastructural evaluation of the radioprotective effects of melatonin against X-ray-induced skin damage in Albino rats

Our knowledge about the radioprotective effects of melatonin against X-ray-induced skin damage is still lacking. To examine these effects, an animal model of 60 Albino rats was used. The animals were divided into five groups: Group 1, nonirradiated; Group 2, X-ray irradiated (XRI, 8 Gy); Group 3, XRI pretreated with solvent (ethanol and phosphate-buffered saline); Group 4, nonirradiated group treated with melatonin; and Group 5, XRI pretreated with melatonin. The skin was evaluated for ultrastructural changes using transmission electron microscopy (TEM). When compared to the nonirradiated skin (Groups 1 and 4), XRI skin (Groups 2 and 3) showed features of both cell injury and increased metabolic activity. The former included changes such as condensation of the nuclei, vacuolization of the cytoplasm, dilatation of the rough endoplasmic reticulum, swelling of the mitochondria with cristolysis, destruction of the ribosomes and intermediate filaments, fragmentation of the keratohyaline granules and loss of the irregularity of the basal cell borders. The central cells of the sebaceous gland alveoli had larger irregular nuclei and few lipid droplets in their cytoplasm. The hair follicle cells had heterochromatic nuclei and less electron dense cytoplasm containing few complements of the organelles. The features of increased metabolic activity included increased euchromatin, irregularity of the nuclear membrane and increased branching of the melanocytes. Also, an increased number of the Birbeck granules were seen in the Langerhans cells. When compared to the irradiated skin (Groups 2 and 3), these changes were mild or absent in the skin of XRI animals pretreated with melatonin (Group 5). The ability of melatonin to minimize the injurious effects of XRI suggests a radioprotective role. The clinical ramifications of these observations warrant further studies.