Safety evaluation of DT388IL3, a diphtheria toxin/interleukin 3 fusion protein, in the cynomolgus monkey

We developed a fusion toxin, DT₃₈₈IL3, consisting of the catalytic and translocation domains of diphtheria toxin (DT₃₈₈) linked to interleukin 3 (IL3) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to estimate a range for the maximum tolerated dose (MTD) and to evaluate the dose-limiting toxicity (DLT) of DT₃₈₈IL3 in cynomolgus monkeys (Macaca fasicularis), which possess cross-reactive IL3 receptors. In our previous study, we administered up to six infusions of DT₃₈₈IL3 at 40, 60, or 100 g/kg every other day to three pairs (one male monkey and one female monkey) of young adult monkeys. In five of six monkeys, results showed a dose-dependent increase in malaise and anorexia but no consistent abnormalities in serum chemistries or blood counts. There was no evidence of organ damage by blood tests or histopathology. However, the female treated at 100 g/kg, died of moderate to severe vasculitis of multiple tissues. Based on these findings, this study repeated the 100 g/kg group and added a group that received 150 g/kg in an effort to confirm a dose response. Two female monkeys were treated with up to six infusions of DT₃₈₈IL3 at 100 g/kg or 150 g/kg every other day. One additional female monkey was treated as a negative control. Monkeys in the 100 g/kg group showed moderate malaise and anorexia, but no consistent abnormalities in blood counts or serum chemistries. Moderate elevations of liver enzymes were noted in the 150 g/kg group in addition to severe malaise and anorexia. No significant findings were revealed at gross necropsy. The histopathological findings revealed regenerative myeloid hyperplasia and hepatic degeneration and regeneration in the 150 g/kg group. Similar lesions of less severity were detected in the 100 g/kg group. DT₃₈₈IL3 plasma half-life was approximately 20 min with a peak concentration of approximately 2 g/ml (30,000 pM). The IC₅₀ for AML blasts in vitro was 6 pM. Collectively, our results suggest that DT₃₈₈IL3 can be tolerated at doses up to 100 g/kg in a nonhuman primate, which is higher than previously reported for other AML directed diphtheria toxin fusion proteins, and should in principle allow for dose escalation with reduced toxic side effects. Based on these findings a phase I clinical trial has recently been initiated with DT₃₈₈IL3 for the treatment of AML.     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel bacterial pathogen (Enterobacter sp.) isolated from the leaf roller, Caloptilia theivora of tea of Darjeeling foothills

Enterobacter sp. was isolated from the diseased and dead caterpillars of the tea leaf roller (Caloptilia theivora) from the Darjeeling foothill region. When the vegetative form of the bacterium was applied via food, mortality of C. theivora showed an LC₅₀ value at 363.1 μg/ml (bacterial wt./vol. of water) with fiducial limits 363.25 and 362.94 μg/ml respectively. The LT₅₀ values for C. theivora were 6 days for 100 μg/ml, 5.96 days for 300 μg/ml, 5.81 days for 500 μg/ml, 4.96 days for 750 μg/ml and 4.61 days for 1,000 μg/ml concentrations. The finding would enable one to contemplate development of a microbial pesticide using this novel Enterobacter sp. DD01 for control of the leaf rolling pest.     

The effects of anhydrous ammonia on membrane stability ofP.hymatotrichum omnivorum

Sclerotia and mycelium ofPhymatotrichum omnivorum were exposed to anhydrous ammonia (NH₃) and then observed with an electron microscope in order to determine the effects of the NH₃ treatment on the fungal membranes. Sclerotia were exposed to four rates of NH₃: 28, 56, 84, and 112μg NH₃/ml of air for 24 hours. At 28μg/ml, the plasmalemma became wavy and the mitochondrial cristae began to swell and disperse. At 56μg NH₃/ml the plasmalemma showed breakage and formation of vesicles, and all other membrane systems within the cell were broken and distorted. All membranes were totally disrupted and no organelles were recognizable at 84μg NH₃/ml. Mycelium was exposed to 2, 4, 8, 20, and 40μg NH₃/ml for one minute. Damage to cell membranes was not observed at NH₃ conc. up to 4μg/ ml. At 8μg NH₃/ml the plasmalemma was broken and the mitochondria were disrupted. At 20μg/ ml and above, all internal organization was destroyed.     

Synthesis and antituberculosis activity of derivatives of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> glycoside steviolbioside and diterpenoid isosteviol containing hydrazone,hydrazide, and pyridinoyl moieties

Conjugates of the antituberculosis drug isoniazid (isonicotinyl hydrazine) and isomeric hydrazides of nicotinic and α-picolinic acid with glycoside steviolbioside from the Stevia rebaudiana plant and the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. In addition, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties joined by a dihydrazide linker were obtained. The parental compounds and their synthetic derivatives were found to inhibit the in vitro growth of Mycobacterium tuberculosis (H₃₇R_V). The measured minimal concentrations of stevio-side and steviolbioside, at which the growth of M. tuberculosis was inhibited by 100% (MIC), were 7.5 and 3.8 μg/ml, respectively. MIC values for steviolbioside and isosteviol conjugates with hydrazides of pyridine carbonic acid were within the ranges of 5–10 and 10–20 μg/ml, respectively. The maximal inhibitory effect against M. tuberculosis was shown by the isosteviol conjugates with adipic acid dihydrazide (MIC 1.7 and 3.1 μg/ml). Antituberculosis activities of the tested compounds were higher than the activity of antituberculosis drug Pyrizanamide (MIC 20 μg/ml) but lower than that of antituberculosis drug isoniazid (MIC 0.02–0.04 μg/ml).     

Protective Effect of <Emphasis Type="Italic">Nigella sativa</Emphasis> Extract and Thymoquinone on Serum/Glucose Deprivation-Induced PC12 Cells Death

The serum/glucose deprivation (SGD)-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Nigella sativa L. (family Ranunculaceae) and its active component thymoquinone (TQ) has been known as a source of antioxidants. In the present study, the protective effects of N. sativa and TQ on cell viability and reactive oxygen species (ROS) production in cultured PC12 cells were investigated under SGD conditions. PC12 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded overnight and then deprived of serum/glucose for 6 and 18 h. Cells were pretreated with different concentrations of N. sativa extract (15.62–250 μg/ml) and TQ (1.17–150 μM) for 2 h. Cell viability was quantitated by MTT assay. Intracellular ROS production was measured by flow cytometry using 2′,7′-dichlorofluorescin diacetate (DCF-DA) as a probe. SGD induced significant cells toxicity after 6, 18, or 24 h (P < 0.001). Pretreatment with N. sativa (15.62–250 μg/ml) and TQ (1.17–37.5 μM) reduced SGD-induced cytotoxicity in PC12 cells after 6 and 18 h. A significant increase in intracellular ROS production was seen following SGD (P < 0.001). N. sativa (250 μg/ml, P < 0.01) and TQ (2.34, 4.68, 9.37 μM, P < 0.01) pretreatment reversed the increased ROS production following ischemic insult. The experimental results suggest that N. sativa extract and TQ protects the PC12 cells against SGD-induced cytotoxicity via antioxidant mechanisms. Our findings might raise the possibility of potential therapeutic application of N. sativa extract and TQ for managing cerebral ischemic and neurodegenerative disorders.     

Rat pituitary tumor cells in serum-free culture. II. Serum factor and thyroid hormone requirements for estrogen-responsive growth

Summary The effect of 17β-estradiol (E₂) on growth of GH₄C₁ rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum. Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E₂ responsive only when growth was retarded by reducing the T₃ concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E₂ to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E₂ to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors. This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc.     

Rat pituitary tumor cells in serum-free culture. I. Selection of thyroid hormone-responsive and autonomous cells

Summary The growth of GH₄C₁, GH₃, GH₁, and GH₃C₁₅ rat pituitary tumor cell lines was studied in a serum-free medium (designated TRM-1) formulated with 1∶1 (vol/vol) mixture of Ham's F12 nutrient mixture and Dulbecco's modified Eagle's medium (F12-DME) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 μg/ml gentamicin supplemented with 10 μg/ml bovine insulin, 10 μg/ml human transferrin (Tf), 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine (Etn), and 500 μg/ml bovine serum albumin. Of the lines evaluated, only the GH₁ failed to grow in TRM-1. Passage of the GH₄C₁ and GH₃ lines from serum-containing medium into TRM-1 caused an initial selection resulting in cells that grew progressively at higher rates and finally were maintained indefinitely in TRM-1. These populations showed a requirement for supraphysiologic concentrations of T₃ (1.0 to 10 nM). After adaptation of the GH₄C₁ line in TRM-1 for ≤20 generations, removal of components gave a less complex mixture containing 15 mM HEPES, 50 μ/ml gentamicin, 10 μg/ml Tf, 10 nM T₃, and 50 μM Etn (designated TRM-2) that supported serial passage of the cells. Under these conditions, thyroid hormone dependence was lost progressively. When T₃ was removed from TRM-2 adapted cells, a third population was selected that no longer required thyroid hormones and was only slightly stimulated by T₃. These studies demonstrated that the combination of serum-containing and serum-free conditions can be used to select pituitary cell populations that a) required both serum-factor(s) and T₃ for optimum growth, b) required supraphysiologic concentrations of T₃ without serum proteins other than Tf and albumin, and c) were completely autonomous in that they proliferated in medium supplemented only with Tf and nutrients without necessity of other serum factor(s) or T₃. This work was supported by grants CA-26617 and CA-38024 from the National Cancer Institute, Bethesda, MD, American Cancer Society grant BC-255, and grant 2225 from the Council for Tobacco Research, Inc., USA.     

Antivibrio compounds produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. W3: characterisation and assessment of their safety to shrimps

In order to explore compounds naturallly inhibitory to shrimp pathogenic vibrios, a culture filtrate of Pseudomonas sp. W3 at a pH of 2 was extracted with ethyl acetate (EtOAc) to produce 82.15 mg/l of a yellow–brown extract (EtOAc-W3) that had MIC values of 225-450 μg/ml against the growth of 18 shrimp pathogenic Vibrio harveyi strains. The MIC of EtOAc-W3 against the most pathogenic strain PSU 2015 was 450 μg/ml and this strain had the lowest LD₅₀ (50% lethal dose) to pacific white shrimp (Litopenaeus vannamei, PL 21). At this MIC value, EtOAc-W3 in artificial sea water (ASW) killed strain PSU 2015; however in natural sea water, only a partial growth inhibition was observed. The toxicity to pacific white shrimp and antivibrio activity of the EtOAc-W3 were investigated by conducting an experiment with 4 sets; native control (commercial ASW), EtOAc-W3 control (MIC/10, 45 μg/ml), challenge (inoculation 6.0 × 10⁶ c.f.u./ml PSU 2015) and treatment (6.0 × 10⁶ c.f.u./ml PSU 2015 + 45 μg/ml EtOAc-W3). The same experiment was repeated by increasing the dose of EtOAc-W3 to 90 μg/ml (MIC/5). Both concentrations of EtOAc-W3 tested had no toxicity to postlarval shrimps. A significant decrease in shrimp mortality was observed over a 72 h period as approximately 80% of the shrimps died in each challenge set but only 63 and 23% died in the presence of 45 and 90 μg/ml EtOAc-W3. The major component of EtOAc-W3 was supposed to be 2-heptyl-4-quinolone (HHQ) by FAB-MS and ¹H-NMR analyses of the purified fraction.     

The effect of dexamethasone on the functioning of C3 receptor sites on the surfaces of human fibroblasts

The presence of C₃ receptor sites on the cell surfaces of WI-38 fibroblasts was reported in a previous paper. Here the effect of dexamethasone sodium sulfate of C₃ receptor site function was studied. The incubation of WI-38 fibroblasts with dexamethasone sodium sulfate produces the biphasic mode of action, i.e., the growth-stimulating phase with low doses (90–230 μg/ml) and the growth-inhibiting phase with high doses (450–900 μg/ml). The function of C₃ receptor sites on WI-38 fibroblasts seems to be very stable and cannot be suppressed by the pretreatment of WI-38 fibroblasts with dexamethasone in high concentrations, where the cell growth is inhibited.     

Extracellular localization of galectin-3 has a deleterious role in joint tissues

In this study we examine the extracellular role of galectin-3 (gal-3) in joint tissues. Following intra-articular injection of gal-3 or vehicle in knee joints of mice, histological evaluation of articular cartilage and subchondral bone was performed. Further studies were then performed using human osteoarthritic (OA) chondrocytes and subchondral bone osteoblasts, in which the effect of gal-3 (0 to 10 μg/ml) was analyzed. Osteoblasts were incubated in the presence of vitamin D₃ (50 nM), which is an inducer of osteocalcin, encoded by an osteoblast terminal differentiation gene. Genes of interest mainly expressed in either chondrocytes or osteoblasts were analyzed with real-time RT-PCR and enzyme immunoassays. Signalling pathways regulating osteocalcin were analyzed in the presence of gal-3. Intra-articular injection of gal-3 induced knee swelling and lesions in both cartilage and subchondral bone. On human OA chondrocytes, gal-3 at 1 μg/ml stimulated ADAMTS-5 expression in chondrocytes and, at higher concentrations (5 and 10 μg/ml), matrix metalloproteinase-3 expression. Experiments performed with osteoblasts showed a weak but bipolar effect on alkaline phosphatase expression: stimulation at 1 μg/ml or inhibition at 10 μg/ml. In the absence of vitamin D₃, type I collagen alpha 1 chain expression was inhibited by 10 μg/ml of gal-3. The vitamin D₃induced osteocalcin was strongly inhibited in a dose-dependent manner in the presence of gal-3, at both the mRNA and protein levels. This inhibition was mainly mediated by phosphatidylinositol-3-kinase. These findings indicate that high levels of extracellular gal-3, which could be encountered locally during the inflammatory process, have deleterious effects in both cartilage and subchondral bone tissues.     

Antioxidant activities of cultured <Emphasis Type="Italic">Armillariella mellea</Emphasis>

This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus—Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition, and FeCl₂-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H₂O) and ethanolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1–100 μg/ml, the free radical scavenging activity was 73.7–92.1% for AM-H₂O, and 60.0–90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC₅₀ is 9.17 μg/ml for AM-H₂O and 7.48 μg/ml for AM-EtOH). In general, AM-H₂O showed a stronger antilipid peroxidation activity on different rat’s tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the antilipid peroxidation activity of AM-H₂O (IC₅₀–6.66 μg/ml) in brain homogenate was as good as IC₅₀–5.42 μg/ml. AM-H₂O (80.0 mg/g) possessed a significantly higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and antilipid peroxidation activities, especially the AM-H₂O in the brain homogenate. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 495–500. The text was submitted by the authors in English.     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: Review of the literature

Voriconazole (Vfend™) is a new triazole that currently is undergoing phase III clinical trials. This review summarizes the published data obtained by NCCLS methods on the in vitro antifungal activity of voriconazole in comparison to itraconazole, amphotericin B, fluconazole, ketoconazole and flucytosine. Voriconazole had fungistatic activity against most yeasts and yeastlike species (minimum inhibitory concentrations [MICs] <2 μg/ml) that was similar or superior to those of fluconazole, amphotericin B, and itraconazole. Against Candida glabrata and C. krusei, voriconazole MIC ranges were 0.03 to 8 and 0.01 to >4 μg/ml, respectively. For four of the six Aspergillus spp. evaluated, voriconazole MICs (< 0.03 to 2 μg/ml) were lower than amphotericin B (0.25 to 4 μg/ml) and similar to itraconazole MICs. Voriconazole fungistatic activity against Fusarium spp. has been variable. Against F. oxysporum and solani, most studies showed MICs ranging from 0.25 to 8 μg/ml. Voriconazole had excellent fungistatic activity against five of the six species of dimorphic fungi evaluated (MIC₉₀s < 1.0 μg/ml). The exception was Sporothrix schenckii (MIC₉₀s and geometric mean MICs ≥ 8 μg/ml). Only amphotericin B had good fungistatic activity against the Zygomycetes species (voriconazole MICs ranged from 2 to >32 μg/ml). Voriconazole showed excellent in vitro activity (MICs < 0.03 to 1.0 μg/ml) against most of the 50 species of dematiaceous fungi tested, but the activity of all the agents was poor against most isolates of Scedosporium prolificans and Phaeoacremonium parasiticum (Phialophora parasitica). Voriconazole had fungicidal activity against most Aspergillus spp., B. dermatitidis, and some dematiaceous fungi. In vitro/in vivo correlations should aid in the interpretation of these results. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sensitivity of selected members of the family Halobacteriaceae to quinolone antimicrobial compounds

Many members of the Halobacteriaceae are inhibited by quinolone compounds, which inhibit type II DNA topoisomerase. Ciprofloxacin was the most potent inhibitor, followed by ofloxacin and norfloxacin. Ciprofloxacin concentrations between 25 and 60 μg/ml caused 50% inhibition of the growth of most Haloferax and Haloarcula species. Halobacterium species were less sensitive. At sublethal concentrations, formation of elongated and/or swollen cells was observed in many species. The alkaliphilic Natronobacterium pharaonis was very sensitive (50% inhibition by ciprofloxacin, ofloxacin, and norfloxacin at concentrations between 4 and 15 μg/ml). The resistance of many members of the Halobacteriaceae to high concentrations of quinolone compounds may in part be due to the high magnesium concentrations present in the growth media. Haloferax volcanii was sensitive to 40 μg/ml ciprofloxacin when grown at suboptimal magnesium concentrations (0.1 M), but was hardly affected by 100 μg/ml of the inhibitor when grown in the presence of 0.5–0.75 M MgCl₂. It is suggested that the putative archaeal type II DNA topoisomerase has properties similar to those of the enzyme from Bacteria, although its sensitivity to quinolone antimicrobial compounds may be lower. Received: 6 November 1995 / Accepted: 26 February 1996     

Growth and fruitbody formation ofGanoderma lucidum on media supplemented with vanadium, selenium and germanium

Responses of mycelia ofGanoderma lucidum to vanadium, selenium and germanium were examined over a wide range of concentrations (10–1, 120 μg/ml) in pure culture. Se and V were found to be highly toxic, but Ge was not toxic at the levels tested.Ganododerma lucidum cultivated on substrates of sawdust with V (30–80 μg/g) developed mature fruitbodies, but the bioaccumulation of V was quite low (2.5–7 μg/g in pileus, 12.5–21.5 μg/g in stipe and <1 μg/g in basidiospores). Se as Na₂SeO₄ labeled with⁷⁵Se was effectively taken up from substrates and accumulated in fruitbodies (mainly in pileus), then depleted by discharge of basidiospores. Ge as GeCl₄ labeled with⁷⁷Ge was easily uptaken and translocated into fruitbodies.     

Effect of α-tocopherol on the synthesis of phosphatidylglycerol catalyzed by phospholipase D in an aqueous system

Phosphatidylglycerol (PG) was synthesized from several phosphatidylcholines (PCs) via phospholipase D (PLD)-catalyzed transphosphatidylation in an aqueous system. The yield of PG were 71 and 68 mol% from soybean PC and egg yolk PC, respectively, under the optimum reaction conditions of 50 μmol PC, 10 mmol glycerol, 3 ml of acetate buffer, 1.6 U PLD, and 30 μmol CaCl₂ at 37°C for 48 h. In case of salmon roe PC with 14.3% eicosapentaenoic acid and 26.8% docosahexaenoic acid, the PG yield increased to 94 mol% by addition of 46 μmol α-tocopherol, although the PG yield was only 10% in absence of α-tocopherol.     

An electrophysiological freeze fracture assessment of cadmium nephrotoxicity in vitro

Summary Human proximal tubule cell cultures exposed to doses of cadmium chloride (CdCl₂) between 0.05 μg/ml and 0.5 μg/ml exhibited alterations in cell membrane structure and transport function. At these Cd concentrations, cell numbers were not significantly altered from control values in either nonreplicating confluent, or actively replicating subconfluent cultures. Transmission electron microscopy revealed few alterations in cultures treated with 0.05 μg/ml Cd. Tight junctions were intact; organelles and myeloid body formation appeared normal. Freeze fracture analysis confirmed the integrity of the tight junctions as well as increased numbers of vesicles or pits along the lateral cell membrane, indicating increased endocytotic activity. Cells exposed to 0.1 μg/ml Cd were characterized by decreased numbers of microvilli and inhibited myeloid body formation. Cd doses of 0.5 μg/ml elicited nuclear chromatin condensation, fragmented sealing strands in 5 to 10% of the tight junction profiles, sparse microvilli, and inhibited myeloid body formation. Electrophysiologic assessments of transport function by Ussing chamber analysis revealed decreases in transepithelial potentials for all three concentrations, with significant differences at Cd concentrations of 0.5 to 0.1 μg/ml. Cells treated with 0.5 μg/ml Cd also exhibited slight decreases in electrical resistance, consistent with the minimal fragmentation of sealing strands observed in freeze fracture replicas. Resistance in cultures treated with 0.1 or 0.05 μg/ml Cd remained within control values and indicated that drops in potential difference and short circuit current in these cells reflected true alterations in ion transport. This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society of Toxicology (SOT) and the Tissue Culture Association field at the 27th annual meeting of the SOT in Dallas, Texas in 1988. This work was supported by the Johns Hopkins Center for Alternatives to Animal Testing. The Balzers Freeze Fracture Unit utilized in these studies was provided by equipment grant S10 RR02329 from the National Institutes of Health, Bethesda, MD.     

Fungicidal effect of hydrogen peroxide on fungal infection of rainbow trout eggs

Exposure to 1,500 μg/ml of hydrogen peroxide (H₂O₂) for 60 min at 13°C was found to be injurious to rainbow trout eggs. On the other hand, the concentration which effectively inhibited pathogenic fungi in vitro was substantially less than this toxic dosage; specifically, 500 μg/ml for 60 min at 20°C to inhibit the zoosporic stage and 1,000 μg/ml for 60 min at 20°C to inhibit the vegetative stage. From in vivo tests, treatment with 1,000 μg/ml of H₂O₂ for 60 min at 13°C was found to be the most effective procedure to control fungal infection and increase the hatching rate of rainbow trout eggs.     

Antimicrobial Butyrolactone I Derivatives from the Ecuadorian Soil Fungus Aspergillus terreus Thorn. var terreus

Summary The fungus Aspergillus terreus Thorn var. terreus isolated from an Ecuador soil sample was cultured in liquid and solid media and yielded three main metabolites identified as terreic acid (1), butyrolactone I (2) and lovastatin (3). The natural products as well as three synthetic butyrolactone I derivatives were assessed for antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi as well as for seed germination and seedling growth. Furthermore, the compounds were assessed as inhibitors towards the enzymes acetylcholinesterase, β-glucosidase, and β-glucuronidase. Terreic acid, butyrolactone I, butyrolactone 4′,4′′-diacetate (2.1), and 3′-(3-methylbutyl)-butyrolactone II (2.2) were active towards the phytopathogenic bacteria Erwinia carotovora with IC₅₀ of 5 and 4–18 μg/ml, respectively. Under the same experimental conditions, the IC₅₀ of streptomycin was 1.9 μg/ml. 3′-(3-Methylbutyl)-butyrolactone II was moderately active against Pseudomonas syringae and Botrytis cinerea with IC₅₀ of 21μg/ml and MIC of 15.6 μg/ml, respectively. Butyrolactone I also inhibited germination of the dicot Lactuca sativa with an IC₅₀ of 5 × 10⁻⁵ M. The IC₅₀ of reference herbicide acetochlor was 1 × 10⁻⁵ M. The effect of 2.2 and 2.3, known as butyrolactone III on Panicum millaceum germination and growth was stronger than that of 2 and 2.1. Reduction of the double bond in the isoprenyl side chain of butyrolactone I increased the antibacterial effect against E. carotovora as well as acetylation. To our best knowledge, this is the first report on the antibacterial effect of butyrolactone derivatives towards Erwinia carotovora and the phytopathogenic fungus Botrytis cinerea. The butyrolactone I derivative 2.2 presented a moderate inhibitory effect against the enzyme acetylcholinesterase with an IC₅₀ of 47 μg/ml. Under the same experimental conditions, the reference inhibitor galanthamine had an IC₅₀ of 3 μg/ml.