Inhibition of in vitro germination and spherulation ofCoccidioides immitis byAllium sativum

An aqueous extract of a dehydrated garlic preparation with uniform consistency inhibited all eight clinical isolates of the dimorphic fungus,Coccidioides immitis. The inhibitory and lethic concentrations were in the range of 3.12–6.25 mg/ml for both the saprophytic (mold) and parasitic (spherule) forms ofC. immitis. At 6.25-mg/ml concentration, the organism lost its viability within 6 h. The conversion of arthroconidia into spherules in a chemically defined liquid medium was prevented by garlic extract diluted to 1:320 (3.12 mg/ml). The data indicate that components of garlic readily inhibited the in vitro germination and spherulation of this medically important dimorphic fungus.     

3H-5-IODO-2'-DEOXYURIDINE TOXICITY PROBLEMS IN CELL PROLIFERATION STUDIES

The toxicity of ³H-5-iodo-2′-deoxyuridine (³H-IUdR) was evaluated by injecting tumor-bearing C3H mice with different concentrations of ethanol (the solvent), different doses of tritium tagged onto either IUdR or thymidine and different chemical doses of IUdR, and then measuring the ³H-IUdR incorporation into duodenal and mammary tumor DNA as well as the cellular kinetics of duodenal crypt cells. Ethanol (37% or less, 0.2 ml/mouse) does not significantly inhibit IUdR incorporation into DNA, and the incorporation after a tritium dose of 75 μCi ³H-IUdR/mouse (about 3 μCi/g body weight) is not less than the incorporation following an injection of 25 μCi ³H-IUdR/mouse when the IUdR dose is below 0.005 μmole per mouse. The toxic effects are primarily due to chemical toxicity from IUdR per se. IUdR, at doses of 0.2 μmoles per mouse does inhibit IUdR incorporation into duodenal and tumor DNA, and the duodenal labeling index and the fraction of labeled mitoses are significantly reduced when 0.013 μmole IUdR per mouse is injected. Also some of the duodenal cells containing IUdR apparently undergo only one post-labeling division and the generation time (T_c) of the cells containing IUdR (25 μCi ³H-IUdR/mouse) is 15.3 hr as compared to 13.3 hr for cells labeled with ³H-T (75 μCi/mouse). This increase in T_c is probably not statistically significant; nevertheless, these results do indicate that one must be exceedingly cautious when using ³H-IUdR as a radiotracer for studies concerned with in vivo cellular kinetics and, at least for C3H mice, the dose should be less than 0.01 μmole per 25 g mouse.     

Coccidioidin sensitivity in control,immunized and infected guinea pigs

Summary Delayed hypersensitivity to potent coccidioidin developed in guinea pigs immunized with deadC. immitis arthrospores and guinea pigs infected with an aerosol ofC. immitis arthrospores at weeks 1 and 2. Delayed hypersensitivity in control animals sensitized by repeated intradermal testing developed at weeks 3 and 4. The delayed hypersensitivity responses were characterized grossly by indurations larger than 25 mm² and could be seen at 6 and 24 hours after testing. Retesting reduced the size of the 24 hour indurations when compared to virginal reactions. The retest delayed reactions in infected animals had indurations at 24 and 48 hours that were larger than those in the other groups.In those animals that were skin test positive but not challenged no tube precipitins, agar gel precipitins, CF antibodies, anaphylaxis or immediate hypersensitivity were detected. Because of the inability to detect precipitins the early phase of the hypersensitivity seen at 6 hours was not considered an Arthus reaction.     

Aluminium-induced morphogenic and biochemical variations ofBacopa Monniera

Shoots and roots ofBacopa monniera (L.) Wettst. have been regenerated from nodal segments on MS medium containing combinations of NAA and BAP. The cultures showed 100% regeneration on MS (sucrose 2%) medium added with NAA (0.2 mg L^-1), BAP (0.5 mg L^-1) and glutamine (50 mg L^-1). Supplemented with aluminium chloride (up to 400 μM), this medium could ensure successful survival of regenerants. AH the regenerants, maintained on AlCl₃-supplemented medium for the last three years, failed to grow when transferred to AlCl₃-free media. Aluminium stress also induced synthesis of proline and proteins. The rate of photosynthesis decreased at increased aluminium concentrations.     

Influence of antimicrobial agents on contamination and chlortetracycline production

The possibility of shortening the thermal sterilization time for cultivating media was demonstrated in chlortetracycline fermentation with an industrial strain ofStreptomyces aureofaciens. The medium was artificially contaminated with a mixture of eight strains of G + and G-bacteria isolated from contaminated industrial fermentors, and the following chemical agents, either alone or in combination, were added: formaldehyde, phenol, dimethylformamide,p-aminosalicylic acid and nitrofurazone. Dimethylformamide was inhibitory even at 0.08%. formaldehyde concentrations higher than 0.05%, Nitrofurazone stimulated chlortetracycline production, The best combination was 0.01% formaldehyde added before, and 2.10^-3% nitrofurazone added after short sterilization at 120 °C.     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.     

Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC₅₀) values of the embryos and larvae were 1.71 and 0.22 mg l⁻¹ for copper and 2.57 and 2.72 mg l⁻¹ for zinc, respectively, whereas the MB LC₅₀ after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l⁻¹, respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l⁻¹ for embryos and 0.03, 0.03 and 1.78 mg l⁻¹ for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l⁻¹, respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l⁻¹ led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.     

Toxicity oft-butylhydroperoxide in hepatocyte monolayers exposed to hypoxia and reoxygenation

Summary Inasmuch as it is known that the toxicity of anesthetic agents is potentiated by hypoxia and that the reductive metabolism of these agents results in the formation of lipid hydroperoxides, we investigated the toxicity of hydroperoxides under low-oxygen concentrations. We found that hypoxia exacerbates the toxicity oft-butyl hydroperoxide, shifting the dose-response curve oft-butyl hydroperoxide vs. lysis of hepatocytes approximately an order of magnitude to the left. Furthermore, although at the end of a 4-h exposure to 0.5% O₂ hepatocyte monolayers seemed normal by three indices (release of⁵¹Cr and serum glutamate transaminase or exclusion of trypan blue), they were completely lysed after an additional 20 h reoxygenation at 20%. O₂. In contrast, monolayers exposed to 2% O₂ for 4 h seemed normal after 20 h reoxygenation. However, cells exposed to both a subtoxic dose of hydroperoxide and 4 h of 2% O₂, although seeming healthy at the end of the hypoxic period, were completely lysed within 20 h after reoxygenation. The study was supported by grant OH 00978 from the National Institutes for Occupational Safety and Health, Atlanta, Georgia.     

Adaptation of stomatal response ofCamellia rusticana to a heavy snowfall environment: Winter drought and net photosynthesis

The adaptation ofCamellia rusticana, an evergreen broad-leaved shrub found in areas of heavy snowfall in Japan, to heavy snowfall environments, and the mechanisms by which it is damaged in winter above the snow, were investigated. The stomatal response and photosynthetic characteristics ofC. rusticana were compared to those ofCamellia japonica found in areas of light snowfall. In field conditions, the mean net photosynthesis ofC. rusticana at photon flux density (PFD) over 200 μmol m⁻²s⁻¹ (Pn(_>200). was 50% larger than that ofC. japonica, but in both light saturated and CO₂ saturated conditions, the O₂ evolution rate (Pc) ofC. rusticana was not different from that ofC. japonica. Mean leaf conductance at PFD over 200 μmol m⁻²s⁻¹ (gl(_>200)) was about 100% larger than that ofC. japonica in the field. The Pn(_>200)) was 50% ratio ofC. rusticana was 37% higher than that ofC. japonica which suggests thatC. rusticana's larger Pn(_>200) can be explained by its larger gl(_>200). WhenC. rusticana trees wintering underneath the snow were projected above it, the leaves of these plants showed serious drought within five days in non-freezing conditions. Their Pc and the maximum stomatal conductance decreased by half and did not recover. The leaves ofC. rusticana showed larger gl(_>200) and a less sensitive stomatal response to the decrease of leaf water potential than that ofC. japonica. The stomata characteristics ofC. rusticana caused larger net photosynthesis than that ofC. japonica during the no snow period, and caused the need for snow cover in winter as protector from winter drought.     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

Metabolite effectors for short-term nitrogen-dependent enhancement of phosphoenolpyruvate carboxylase activity and decrease of net sucrose synthesis in wheat leaves

It has been demonstrated previously that field pea (Pisum sativum L. cv. Express) grown in hydroponic culture on a complete nutrient solution with low NH₄⁺ concentrations (<0.5 mM) will produce a larger than normal proliferation of nodules. Peas grown in the absence of mineral N in hydroponic culture have been shown to rapidly autoregulate nodulation, forming a static nodule number by 14 to 21 days after planting. The present study further characterizes the effect of NH₄⁺ concentration in hydroponic culture on nodulation and nodule growth. Peas were grown continually for 4 weeks at NH₄⁺ concentrations that were autoregulatory (0.0 mM), stimulatory (0.2 mM) or inhibitory (1.0 mM), or peas were transferred between autoregulatory or NH₄⁺ inhibited and stimulatory solutions after 2 weeks. The peas nodulated as expected when grown under constant autoregulatory, stimulatory or inhibitory concentrations of NH₄⁺. When peas were transferred from the inhibitory (1.0 mM) to the stimulatory solution (0.2 mM) a massive proliferation of nodule primordia over the entire root system was observed within 3 days of the transfer. When they were transferred from the autoregulatory (0.0 mM) to the stimulatory (0.2 mM) solution a 10-day delay occurred before a proliferation in nodule primordia occurred at distal regions of the root system. These findings support our hypothesis that low concentrations (<1.0 mM) of NH₄⁺ in hydroponic culture cause a suppression of autoregulation in pea. In addition, the temporal and spatial differences in nodule proliferation between transfer treatments demonstrate at a whole plant level that autoregulation and NH₄⁺ inhibition suppress early nodule development via different mechanisms.     

Ubiquinone system,GC contents and cellular fatty acid composition of species of the form-genusMalbranchea andCoccidioides immitis for chemotaxonomic study

Ubiquinone system, GC contents and cellular fatty acid (CFA) composition were analyzed as criteria for chemotaxonomy ofMalbranchea species andCoccidioides immitis, which are suggested to be phylogenetically related. Based on the major ubiquinone,Malbranchea spp. were divided into two groups, of which one group possessed the same major ubiquinone asC. immitis. Similar GC contents and CFA profiles were obtained for the species ofMalbranchea andC. immitis. On the basis of these criteria the relationships between the fungi are discussed.     

Pharmacokinetics of anti-ganglioside GD2 mAb 14G2a in a phase I trial in pediatric cancer patients

A phase I trial of a murine anti-ganglioside (GD2) monoclonal antibody (mAb) 14G2a was conducted in 14 neuroblastoma patients and 1 osteosarcoma patient to assess its safety, toxicity and pharmacokinetics in pediatric patients. The pharmacokinetics of mAb 14G2a were biphasic with at ₁ ^2/ of 2.8±2.8 h and at ₁ ^2/ of 18.3±11.8 h. In general,t ₁ ^2/ was dose-dependent with a level of significance ofP=0.036, and it reached a plateau at doses of 250 mg/m² or more. Overall the peak serum levels were dose-dependent atP<0.001. However, they demonstrated an abrupt increase between doses of 100 mg/m² and 250 mg/m². The latter two suggest a saturable mechanism for mAb elimination. In addition, peak serum concentrations were observed earlier at higher mAb doses, which indicates the achievement of a steady state. Thet ₁ ^2/ of mAb 14G2a in children appears to be shorter than in adults. Furthermore, 2 patients demonstrated a considerable decrease int ₁ ^2/ following retreatment with 14G2a. This was paralleled by high human anti-(mouse Ig) antibody levels. This study represents the first comprehensive analysis of murine mAb pharmacokinetics in children and will be useful in the future design of mAb therapy.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439 and in part by a grant from the general Clinical Research Center program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M. M. U.-F. and C.-S. H. were supported in part by a grant from the Children's Cancer Research Foundation, and R. A. R. was supported in part by NIH grant CA 42508     

Membranous localization and properties of ATPase of rat liver lysosomes

Summary Lysosomes isolated from rat liver were found to have ATPase activity (EC No. 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2mm ATP and is inhibited by high concentrations of ATP. The apparentK _m for divalent metal is 0.2mm, and either ca²⁺ or Mg²⁺ give maximal activity.The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity ofL fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosamindase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.     

Environmental Stress Determines the Quality of Bacterial Lysate and Its Utilization Efficiency in a Simple Microbial Loop

Heterotrophic bacteria provide the critical link in the microbial loop by converting dissolved organic matter (DOM) into particulate form. In this study, DOM was prepared from recently isolated estuarine bacterial strain Vibrio sp. (DSM14379) grown at different salinities [0.2%, 0.5%, 3%, 5%, or 10% (w/v)], washed, concentrated, and lysed by autoclaving. The corresponding lysate-containing media were designated LM_0.2, LM_0.5, LM₃, LM₅, and LM₁₀. Vibrio sp. cells grown at different salinities had similar C/N/P ratios, but different C/S ratios, different trace element composition, and different 2D gel electrophoresis protein profiles. Pseudoalteromonas sp. (DSM06238) isolated from a similar environment was able to grow on all lysates, and its biomass production was dependent on lysate type. The highest growth rate and biomass production of Pseudoalteromonas sp. at saturation lysate concentrations were observed in LM₃. The biomass production at saturation lysate concentrations was about 3-fold higher as compared to LM_0.2 and LM₁₀. The initial respiration rate, intracellular adenosine triphosphate (ATP) levels, and ³H-Leu and ³H-TdR incorporation rates were lowest in LM₃. On the other hand, in LM_0.2 or LM₁₀ lysates the situation was reversed, the growth rates and biomass production were lowest, whereas ³H-Leu and ³H-TdR incorporation, respiration rates, as well as ATP levels, were highest. These results imply uncoupling of catabolism from growth in either high- or low-salinity lysates. The results also suggest that differences in organic carbon quality generated during Vibrio sp. growth at different NaCl concentrations were propagated through the simple microbial loop, which may have important ecological implications for higher trophic levels that depend on microbial grazing.     

Effects of butylated hydroxyanisole,butylated hydroxytoluene and tertiary butylhydroquinone on growth and luteoskyrin production byPenicillium islandicum

Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) alone in cultural media were tested for the inhibition of growth and luteoskyrin production by two toxigenic strains ofPenicillium islandicum UST-11 andP. islandicum HLT-6. In potato dextrose agar, the concentrations of BHA and TBHQ from 0.2 mg/disc, BHT from 5.0 mg/disc did affect the growth of both tested strains, but the initial concentrations of these antioxidants to reduced luteoskyrin production by UST-11 strain were BHA 0.5 mg/disc, BHT 1.0 mg/disc and TBHQ 0.4 mg/disc, while for HLT-6, BHA 0.4 mg/disc, BHT and TBHQ were 0.2 mg/disc, respectively. In grainy and powdery rice media, the effects of BHA, BHT and TBHQ on luteoskyrin production byP. islandicum UST-11 and HLT-6 were clearly demonstrated. The efficiency of the inhibitory effect was not only closely related to the concentration of antioxidants, but also completely inhibited the luteoskyrin production at a concentration of 200 mg/kg or higher. Also, the antioxidants at a concentration higher than 20 mg/kg reduced significantly the growth and luteoskyrin production by both strains ofP. islandicum.     

Conversion of Coccidioides immitis in tissue culture

Summary Utilization of a pre-treatment resulted in the establishment of a simple technique to enhance spherulation ofC. immitis in tissue culture.Establishment of the fact that culture spherules are produced from mycelial elements was also recognized and should be included in the life cycle ofC. immitis when cultured in vitro.     

The role of root-associatedKlebsiella pneumoniae in the nitrogen nutrition ofPoa pratensis andTriticum aestivum as estimated by the method of15N isotope dilution

Summary The technique of¹⁵N isotope dilution was used to verify that nitrogen was fixed and transferred to the plant byKlebsiella pneumoniae strain Pp in association withPoa pratensis orTriticum aestivum. Surface sterilized, sprouting seeds were inoculated withK. pneumoniae and grown in sand in modified Leonard jars. Potassium nitrate enriched with¹⁵N was used to provide N concentrations ranging from 10–40 mg Nl^–1 nutrient solution. After 10–18 weeks the shoots and roots were analyzed separately for dry matter, N content, total N, and atom %¹⁵N excess. The acetylene reduction technique was used to test for the presence of N₂-fixing organisms on the roots. The data from¹⁵N isotope dilution demonstrated that up to 33.8% of N in the shoots ofP. pratensis and 15.9% in those ofT. aestivum were derived from associative N₂ fixation byK. pneumoniae. In most experiments the dry matter yield, N content, and total N yield of the shoots ofP. pratensis were increased byK. pneumoniae inoculation, whereas inoculation had no significant effect on the dry matter yield, N content or total N of the shoots ofT. aestivum.     

Effects of temperature,ph, salinity,and inorganic nitrogen on the rate of ammonium oxidation by nitrifiers isolated from wetland environments

Ammonium-oxidizing bacteria were examined in two wetland environments, a freshwater marsh and an estuarine bay, during a 2-year period. Two predominant types were consistently isolated, one from each environment. Both isolates were identified as species ofNitrosomonas. Using a closed culture, high cell density assay, the effects of temperature, pH, salinity, Na⁺, K⁺, nitrite, nitrate, and ammonium concentrations on ammonium oxidation were determined. Maximum activity was observed for the freshwater isolate at 35°C, pH 8.5, salinities of 0.3 to 0.5% Na⁺ and K⁺, and ammonium concentrations greater than 0.5 g/l. For the estuarine isolate, maximum activity was observed at 40°C, pH 8.0, salinities of 0.5 to 1.0%, 1.0% Na⁺ and K⁺, and 0.2 g/l ammonium. The estuarine isolate had a Na⁺ requirement which could be partially substituted by the K⁺, suggesting that the organism is a true estuarine bacterium. Nitrite inhibited both isolates at concentrations greater than 5 mg/l, whereas nitrate had no significant effect on either isolate.     

Toxicity of chromium to the marine planktonic copepodAcartia clausi,Giesbrecht

The toxicity of chromium to the marine planktonic copepodAcartia clausi, Giesbrecht was studied. The LC50 48 h values (concentration of chromium lethal to 50% of the test animals) vary with the experimental temperature, the form of chromium compound tested and the annual generation to which theAcartia specimens belong. The elevation of temperature resulted in a considerable increase ofAcartia's sensitivity to chromium. Cr⁶⁺ in the form of Na₂CrO₄ was more toxic toA. clausi than in the form of CrO₃ · Cr³⁺ in the form of Cr(NO₃)₃ 9 H₂O precipitated to the bottom and was not toxic toAcartia.Acartia specimens belonging to the summer generation were more sensitive to chromium than those belonging to the winter or autumn generation. The exposure ofAcartia to sublethal concentrations of chromium resulted in a reduction of its longevity proportional to the chromium concentrations used.Furthermore, whenA. clausi was exposed to sublethal chromium concentrations it showed a decrease of feeding capacity and increase of respiratory rates, which became more pronounced with increasing chromium concentrations.