Plant regeneration from hypocotyl segments of Lupinus luteus cv. L. Aurea

Complete plants of Lupinus luteus L. cv. Aurea that were regenerated from hypocotyl segments, bloomed, produced seeds and were efficiently nodulated by Bradyrhizobium sp. strains. The highest rates of shoot formation were obtained on A medium plus 1.3% agar with 10.0 M 2-isopentenyladenine (2iP) and 0.11 M naphthaleneacetic acid (NAA); the best rooting was achieved on a medium with 0.5 M NAA plus 0.05 M 2iP. Afterward, plantlets were transferred to either perlite or peat-containing pots and irrigated with a N-free nutrient solution until maturity. Direct rooting of hypocotyls could also be obtained on A medium with 1% agar.     

Method of isolation of cysteine constitutive mutants of the cysteine regulon in Salmonella typhimurium.

Summary Size and shape of mitochondrial DNA molecules of Schizosaccharomyces pombe were analyzed by electron microscopy. Besides numerous linear molecules, circular molecules ranging from 0.83 m to 12.81 m were found. Depending on the method of preparation, both closed and open circular molecules were found. Most of the circular molecules could be assigned to five major size classes of 0.83±0.05 m, 1.7±0.05 m, 4.74±0.04 m, 5.74±0.04 m, and 8.32±0.07 m. Possible explanations for the different size classes of mitochondrial DNA molecules are discussed.     

6-Methylpurine-induced inhibition of sclerotia morphogenesis in Sclerotium rolfsii and its reversal by adenosine

In liquid synthetic medium inoculated with Sclerotium rolfsii (SR), addition of 6-methylpurine (MP, 50g/ml) immediately after inoculation led to approximately 100% reduction in sclerotia production. Adenosine, and to a lesser extent guanosine, each at final concentration of 100g/ml significantly reduced inhibition of sclerotia formation by SR in presence of 50g/ml MP. Uridine and cytidine each at 100g/ml had no such effect. The inhibition of sclerotia morphogenesis could be prevented by addition of 800g/ml of adenosine together with 50g/ml MP. Reversal by adenosine of MP-induced inhibition of sclerotia development was concentration dependent.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Effects of lead and cadmium on chemical composition and total water content of the pupal parasitoid, Pimpla turionellae

The parasitoid Pimpla turionellae L. (Hymenoptera, Ichneumonidae) was fed on Cd, Pb and Cd+Pb-contaminated food (33g Cd, 82g Pb and 33g Cd+82g Pb per gram food fresh weight, respectively). Significant decrease in the total lipid and protein content was found along with an increase in the water content particularly in Cd-contaminated parasitoids.     

Effects of trichloroethylene,tetrachloroethylene and dichloromethane on enzymatic activities in soil

Summary Enzyme assays for -glucosidase, -acetylglucosaminidase, phosphatase, phosphodiesterase, and proteinase were made in soil samples incubated for two months after contamination with trichloroethylene, tetrachloroethylene, and dichloromethane. These volatile chlorinated hydrocarbons were added at doses of 10, 100, and 1000 g per 100 g dry soil, respectively. Almost no effect was observed in soil sample contaminated with 10 g of the chemicals when compared with control soil. When 100 g of the volatile chlorinated hydrocarbons was added, the activity of -glucosidase, -acetylglucosaminidase and, in part, also of proteinase, was reduced during the first 28 days of incubation but returned to the same or slightly higher level than in the control soil after 2 months. Trichloroethylene, tetrachloroethylene, and dichloromethane at a concentration of 1000 g per 100 g soil primarily inhibited activity of all enzymes under test. However, after two months, the enzymatic activities especially in soil samples contaminated with tetrachloroethylene and dichloromethane were found to be at the same or higher level than in the control soil.     

Aflatoxin influences on seed germination and root elongation by two cultivars of Glycine max and uptake of 65 Zn-ZnCl2 by the cultivars

Maximum inhibition of Glycine max, cv. Essex seed germination occurred at 10 g/ml following 72 hr imbibition in constant light. Seeds imbided 108 hr in constant darkness at this concentration showed a 20% rise in germination over that of the control. Imbibition of G. max, cv. Williams seeds in either light or dark for 96 hr did not suppress germination. Imbibition of Essex seeds in either light or dark at 2.5 through 10 g/ml stimulated root elongation except for 10 g/ml at 96 hr (light). Maximum inhibition of Williams root elongation under constant light was at 48 and 72 hr with 10 g/ml. Statistically significant differences in cotyledon, leaf and stem lengths between non-treated (NT) and treated (T) seedlings were not found except for Williams stem length at 2.5 / ml. Root elongation was stimulated 1.2- and 1.1-folds, respectively, at 5.0 (Essex) and 2.5 (Williams) g/ml. Toxin at 2.5 through 10.0 g/ml did not markedly alter either cotyledon or leaf widths with the exception of Williams leaf width at 2.5 g/ml. Medium supplementation with 2.5 through 10.0 g/ml resulted in cotyledon, leaf and root weight enhancements for Essex seedlings. Stem weight was not markedly affected. An 18% rise in Williams cotyledon weight above that of the control was seen at 2.5 g/ml. Williams leaf weights were increased 1.75- and 1.25-folds, respectively, at 2.5 and 10.0 g/ml. Aflatoxin B₁, at 2.5 g/ml promoted Williams stem and root elongation 1.20- and 1.09-folds, respectively. Most of the radioactivity from ⁶⁵Zn-ZnCl₂ recovered within organs was found within Essex roots for both T and NT seedlings. A higher amount of radioactivity was recovered within roots at each toxin concentration than was without toxin. However, this was not statistically significant. Significant differences in the distribution of radioactivity within roots between NT and T Williams seedlings were not observed. Generally, AFB₁ failed to affect significantly these two varieties of soybeans based on the tests relating to germination, growth and radiolabel uptake.     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

Gangliosides Attenuate Ethanol-Induced Apoptosis in Rat Cerebellar Granule Neurons

Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on culture in a serum-free medium containing a depolarizing concentration of KCl (25 mM), 5 M MK-801 (an NMDA receptor antagonist), and 20–200 mM ethanol for 1–4 days. Cell death augmented by ethanol was concentration- and time-dependent with neurons displaying hallmark apoptotic morphology and DNA fragmentation that correlated with the activation of cytosolic caspase-3. Inclusion of 5 M MK-801 or 100 M glycine in culture media did not alter rates of cell death indicating ethanol toxicity is mediated via an NMDA receptor-independent pathway. Preincubation with 50 M gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 M LIGA₂₀ (a semisynthetic GM1 with N-dichloroacetylsphingosine) for 10 min, attenuated caspase-3 activity and ethanol-induced cell death. Data show native gangliosides and a synthetic derivative are potently neuroprotective in this model of ethanol toxicity, and potentially serve as useful probes to further unravel the mechanisms relevant to neuronal apoptosis.     

The initiation of callus culture ofMichelia champaca for essential oil production

Summary TheMichelia champaca callus was induced from rachises of theM. champaca flowers on 1/2 MS medium containing 3% sugar and 0.9% agar. The medium was also supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) as the growth regulators. It was observed that the calli ofM. champaca could be induced on media containing 0–50 M 2,4-D and 0.1–1 M BAP. The calli were grown well on media on media containing 0.1–1.0 M BAP and 2,4-D up to 50 M. As 1 M BAP was used, a lower concentration of 2,4-D was associated with a fast initiation of calli, but the culture of these calli turned brown quickly. To the contrary, a higher concentration of 2,4-D led to a slower rate of callus formation and the culture hardly turned brown. The optimum pH for the cell culture was about 5.6 as 1 M of 2,4-D and BAP were present in the medium.     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

The relationship between cell size and viability of soil bacteria

The number of bacterial cells in soil that form colonies on nutrient agar represent a small fraction of the direct microscopic counts (DMC). The colony-forming cells have larger cell dimensions than the very small (dwarf) cells which represent the majority of the DMC. This may indicate that the dwarf cells are species unable to form visible colonies on agar, or that they swell to normal dimensions when growing. Indigenous bacterial cells were separated from soil by density gradient centrifugation and fractionated according to diameter by filtration through polycarbonate filters. Each filtrate was studied with respect to DMC, cell dimensions, colony-forming cells (visible colonies and microcolonies), and cell dimensions during growth on the agar. The calculated average percent viability was only 0.2% for cells with diameters below 0.4m, about 10% for cells with diameters between 0.4 and 0.6m, and 30–40% for cells with diameters above 0.6m. Only 10–20% of the viable cells with diameters <0.4m increased their diameter to >0.4m prior to growth. Thus, size change during starvation and growth cycles did not explain the high numbers of dwarf cells observed by microscopy. The results show that despite the relatively low number of colony-forming bacteria in soil, the species that form colonies may be fairly representative for the medium size and large cells, which constitute a major part of the bacterial biovolume. Thus plate counting could be a useful method to count and isolate the bacteria accounting for much of the biovolume in soil. The origin of the dwarf cells is still unclear, but the low number of small cells that increased in size seems to indicate that the majority of these bacterial cells are not small forms of ordinary sized bacteria.     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

Influence of nitrogen on the behaviour of nickel in wheat

A glasshouse experiment was conducted to study the effect of Ni on the growth and nutrients concentration in wheat (Triticum aestivum Cv. WH 291) in the presence and absence of applied N as urea. Responses to N application were observed up to 120 g N g^–1 soil. No response to Ni was observed in the dry matter yield of wheat tops (leaves + stem) in the absence of applied N while in the presence of applied N, significant yield increases were obtained at 12.5g Ni g^–1 soil. Nickel was not toxic to wheat up to 50g Ni g^–1 soil in the presence of 120g N g^–1 soil. Nitrogen and Ni concentration in wheat tops and roots increased with increasing levels of applied N and Ni, respectively. Applied Ni had an antagonistic effect on N concentration. Similarly, N reduced the Ni concentration in the wheat tissues. Positive growth responses to Ni were associated with 22 and 15g Ni g^–1 in wheat tops, in the presence of applied N at 60 and 120g N g^–1 soil, while Ni toxicity was associated with 63, 92.5 and 112.5g Ni g^–1 in wheat tops, in the absence and presence of applied N at 60 and 120g N g^–1 soil, respectively.     

Effect of 2-hydroxybenzoate on the rate of naphthalene mineralization in soil

The effect of 2-hydroxybenzoate (2-OHB, salicylate) on the mineralization rate of [¹⁴C]naphthalene, the population density of naphthalene-degrading bacteria, and the concentration of genes encoding for naphthalene dioxygenase in a soil bacterial community was investigated. Six different concentrations of 2-OHB (10, 20, 50, 100, 150 and 200 g g^–1 soil) were tested in 100-g portions of soil. The addition of 10, 20 or 50 g 2-OHB g^–1 soil produced a general increase in total soil bacterial population density, whereas the addition of 100 g or 200 g 2-OHB g^–1 soil specifically increased the proportion of naphthalene degraders relative to the total population. The addition of 50 g 2-OHB g^–1 soil produced a fourfold increase (the maximum observed) in the rate of naphthalene mineralization relative to the rate in unamended soil. The concentration of 2-OHB ( 100 g/g) added to soil correlated with the population density of naphthalene degraders (r=0.961). Addition of up to 200 g 2-OHB g^–1 correlated with the abundance of DNA sequences homologous to known naphthalene dioxygenase genes (nahAB) (r=0.958). However, mineralization of [¹⁴C]naphthalene was stimulated significantly only by the addition of 50 g 2-OHB g^–1 soil. Results of the mineralization experiments were supported by the detection of nahAB mRNA extracted directly from soil. The specificity of the effect of 2-OHB on naphthalene biodegradation was confirmed in a control experiment using equivalent concentrations of 4-OHB which repressed naphthalene mineralization by about 50%. Addition of ammonium nitrate to the soil also increased the rate of naphthalene mineralization. Ammonium nitrate added together with 2-OHB reduced the mineralization enhancement effect of either compound alone. The study confirmed that specific induction of biodegradative genes can enhance chemical pollutant removal in situ. Correspondence to: O. A. Ogunseitan     

Susceptibility of clinical isolates of yeasts to anti-fungal agents

The antimicrobial activity of amphotericin B, 5-fluorocytosine, nystatin, clotrimazole and miconazole were compared in vitro against 244 strains of yeasts that had been isolated from clinical specimens. The yeasts used in this study included 20 species of Candida, Cryptococcus, Saccharomyces Geotrichum, Rhodotorula, Torulopsis and Trichosporon. The majority of the strains (78%) had an MIC of 0.5 g/ml for amphotericin B, 81% an MIC of 1 g/ml for 5-fluorocytosine, 99% 8 g/ml for nystatin, 91%, 8.0 g/ml for clotrimazole and 98% had an MIC of 4.0 for miconazole. Of the anti-fungal agents tested, 5-fluorocytosine and nystatin were found to have the greatest antifungal activity.     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

The bactericidal effects of Lactobacillus acidophilus,garcinol and Protykin® compared to clarithromycin,on Helicobacter pylori

Chronic infection with Helicobacter pylori causes peptic ulcers, gastric cancer and lymphoma. We evaluated the inhibitory effects of the probiotic Lactobacillus acidophilus DDS-1J, the antibiotic clarithromycin and the natural antioxidants garcinol and Protykin^® (containing 50% trans-resveratrol) on Helicobacter pylori strain ATCC 49503. The findings of this study indicate that Lactobacillus acidophilus DDS-1J exerts a growth inhibitory effect on H. pylori at a ratio of 1:1 or higher in vitro. In the case of clarithromycin, garcinol and resveratrol, the bactericidal effect is time and concentration dependent. Clarithromycin completely inhibited growth at 62.5 g/ml at 6 h and at 31.5 g/ml at 12 h. For garcinol the highest concentration needed for complete inhibition was 31.5 g/ml at 6 h and 3.9 g/ml after 12 h incubation. For resveratrol, significant inhibition was noted at 1000 g/ml at 12 h only. The bactericidal effect of garcinol was reduced by the addition of resveratrol at all concentrations 125 g/ml at 6 and 12 h. We conclude from this study that Lactobacillus acidophilus DDS-1J inhibits H. pylori at 1:1 and higher ratios. Also, between the two antioxidants, garcinol is much more potent than resveratrol as a bactericidal agent against H. pylori, and that resveratrol may antagonize this effect. Finally, our study showed equivalent or better bactericidal activity of garcinol compared to clarithromycin against H. pylori at 6 and 12 h incubation, indicating a potential role for this antioxidant in treatment for H. pylori infection.     

Plantlet regeneration from cultured leaves of Cydonia oblonga L. (quince)

Summary Adventitious shoots of Cydonia oblonga Quince A were obtained from leaves cultured on MS-N6 medium containing thidiazuron (TDZ) and -naphthaleneacetic acid (NAA). The frequency of regeneration was high (78% of the cultured leaves with 3.2 shoots per regenerating leaf) at 32 M TDZ plus 0.3 M NAA on young leaves obtained from micropropagated shoots. Shoots were rooted by culturing them first on medium containing 5 M NAA for one week and then on auxinfree medium for four weeks. The regeneration protocol may be useful for selection of somaclonal variants with increased tolerance to low Fe and for transformation mediated by Agrobacterium.