Effect of Ceramide on Mesenchymal Stem Cell Differentiation Toward Adipocytes

Proinflammatory cytokines such as tumor necrosis factor (TNF) α are well known to inhibit adipocyte differentiation. TNF-α triggers ceramide synthesis through binding of TNF-α to its p55 receptor. Therefore, ceramide is implicated in many of the multiple signaling pathways initiated by TNF-α. In breast tissue engineering, it is important to know how to modulate adipocyte differentiation of the stem cells with exogenous additives like ceramide in vitro. We hypothesized that stem cell adipogenesis could be retained in TNF-α-treated preadipocytes in which ceramide synthesis was blocked and that exogenous ceramide could inhibit adipocyte differentiation. We first studied the effect of ceramide synthase inhibitor, Fumonisin B2, on the adipogenesis of murine mesenchymal stem cells (D1 cells), treated with TNF-α. We then studied the effect of specific exogenous C6-ceramide on D1 cell viability and differentiation. It was found that 1 ng/ml of TNF-α significantly inhibited D1 cell adipogenesis. Cells treated with 5 μM of Fumonisin B2 were able to undergo adipogenesis, even when treated with TNF-α. High concentrations of exogenous C6-ceramide (>50 μM) had an inhibitory effect, not only on the pre-confluent proliferation of the D1 cells but also on the post-confluent cell viability. High concentrations of C6-ceramide (>50 μM) also inhibited mitotic clonal expansion when D1 cell differentiation was induced by the addition of an adipogenic hormonal cocktail. C6-ceramide at low concentrations (10–25 μM) inhibited lipid production in D1 cells, demonstrated by decreased levels of both total triglyceride content and specific fatty acid composition percentages. Genetic expression of peroxisome proliferator-activated receptor (PPAR) γ and aP2 in D1 cells was reduced by C6-ceramide treatment. CCAAT/enhancer-binding protein (C/EBP) β levels in D1 cells were reduced by C6-ceramide treatment during early differentiation; PPARγ and aP2 protein levels were reduced at terminal differentiation. C6-ceramide at lower concentrations also decreased lipid accumulation of differentiating D1 cells. Our results suggest that ceramide synthase inhibitor retains the adipogenic potential of TNF-α-treated mesenchymal stem cells, while exogenous ceramide at lower concentrations inhibit the adipogenesis of mesenchymal stem cells. Ceramide, therefore, could be a modulator candidate in breast tissue engineering strategies.     

Development of DNA-Designed Avian IgY Antibodies for Quantitative Determination of Bovine Interferon-Gamma

Interferon-gamma (IFN-γ), a cytokine produced by sensitized T lymphocytes, is one of the key elements in defining T helper 1 lymphocyte immune responses. Quantitative evaluation of IFN-γ expression could provide an important analytical tool for measurement of cell-mediated immunity and investigating immune responses to infectious diseases. Method of DNA-designed avian IgY antibodies was used for production of monospecific polyclonal antibodies that allows quantification of the recombinant bovine IFN-γ protein. IFN-γ cDNA was subcloned and expressed in mammalian expression plasmid (pcDNA3.1(+)) under the control of the human cytomegalovirus promoter. Chickens were immunized by plasmid DNA, and eggyolk antibodies extracted from eggs were collected after immunization. IgY-specific antibodies were evaluated by an antigen capture enzyme-linked immunosorbent assay (ELISA) using recombinant IFN-γ. Based on the results, developed bovine IFN-γ capture ELISA could detect up to 1 ng/ml of IFN-γ by 64-fold diluted IgY. Monospecific anti-bovine IFN-γ antibodies generated in chickens are useful for quantifying different concentrations of recombinant bovine IFN-γ, which is expressed in cell culture.     

Characterization and functionalization of cellulose microbeads for extracorporeal blood purification

In the frame of this work, cellulose microbeads with an average particle size of 2.3 μm were characterized with respect to porosity using a batch solute exclusion method and two groups of model substances, namely proteins and polystyrene sulfonates. The pores of the microbeads were almost completely accessible to proteins with Stokes radii below 2.5 nm. More than 60% of the pores were accessible to albumin, which is relevant for the application in blood purification, since many target substances are albumin bound. Activation of the microbeads with increasing amounts of sodium metaperiodate yielded matrices with dialdehyde contents between 100 and 1,000 μmol/g. The activated beads were well suited for the covalent attachment of functional ligands, such as antibodies. Immobilization of antibodies against the pro-inflammatory cytokine TNF-α resulted in efficient TNF-α adsorbents which possess application potential in extracorporeal blood purification, e.g. for the modulation of cytokine levels as supportive therapy for sepsis and other inflammatory disorders.     

Suitability of ultra-high performance liquid chromatography for the determination of fat-soluble nutritional status (vitamins A, E, D, and individual carotenoids)

Our aim was to assess the suitability of ultra-high performance liquid chromatography (UHPLC) for the simultaneous determination of biomarkers of vitamins A (retinol, retinyl esters), E (α- and γ-tocopherol), D (25-OH-vitamin D), and the major carotenoids in human serum to be used in clinical practice. UHPLC analysis was performed on HSS T3 column (2.1 × 100 mm; 1.8 μm) using gradient elution and UV–VIS detection. The system allows the simultaneous determination of retinol, retinyl palmitate, 25-OH-vitamin D, α- and γ-tocopherol, lutein plus zeaxanthin, α-carotene, β-carotene, α- and β-cryptoxanthin and lycopene. The method showed a good linearity over the physiological range with an adequate accuracy in samples from quality control programs. Suitability of the method in clinical practice was tested by analyzing samples (n = 286) from patients. In conclusion, UHPLC constitutes a reliable approach for nutrient/biomarker profiling allowing the rapid, simultaneous and low-cost determination of vitamins A, E, and D (including vitamers and ester forms) and the major carotenoids in clinical practice.     

A Superoxide Dismutase Purified from the Rhizome of <Emphasis Type="Italic">Curcuma aeruginosa</Emphasis> Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K _m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V _max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min⁻¹ mg protein⁻¹. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn²⁺and Fe²⁺ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC₅₀ = 14.36 ± 0.15 μg protein/ml).     

Folate analysis in foods by UPLC-MS/MS: development and validation of a novel, high throughput quantitative assay; folate levels determined in Australian fortified breads

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed, optimised and validated for the quantification of synthetic folic acid (FA), also called pteroyl-l-glutamic acid or vitamin B9 and naturally occurring 5-methyltetrahydrofolate (5-MTHF) found in folate-fortified breads. Optimised sample preparation prior to analysis involved addition of ¹³C₅ labelled internal standards, treatments with α-amylase and rat serum, solid-phase extraction using aromatic-selective cartridges and ultra-filtration. Analytes were separated on a Waters ACQUITY HSS T3 column during a 6-min run and analysed by positive ion electrospray selected reaction monitoring MS/MS. Standard calibration curves for the two analytes were linear over the range of 0.018–14 μg FA/g of fresh bread (r ² = 0.997) and 9.3–900 ng 5-MTHF/g of fresh bread (r ² = 0.999). The absolute recoveries were 90% and 76% for FA and 5-MTHF, respectively. Intra-day coefficients of variation were 3% for FA and 18% for 5-MTHF. The limit of detection was 9.0 ng/g for FA and 4.3 ng/g for 5-MTHF, determined using pre-extracted tapioca starch as the blank matrix. The assay is rugged, fast, accurate and sensitive, applicable to a variety of food matrices and is capable of the detection and quantification of the naturally occurring low levels of 5-MTHF in wheat breads. The findings of this study revealed that the FA range in Australian fortified breads was 79–110 μg/100 g of fresh bread and suggest that the flour may not have the mandated FA fortification level (200–300 μg/100 g of flour), though this cannot be determined conclusively from experimental bread data alone, as variable baking losses have been documented by other authors.     

The Correlations Between TCA-Glyoxalate Metabolite and Antibiotic Production of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M4018 Grown in Glycerol,Glucose, and Starch Mediums

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5–20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.     

A neutral polysaccharide from <Emphasis Type="Italic">Glycyrrhiza inflata</Emphasis>

A neutral polysaccharide Gi-A1 was isolated from the roots of Glycyrrhiza inflata Bat. It had a molecular mass of over 2000 kDa and showed [α]_D²⁰ + 81.4° (c 1.05, H₂O). Acid hydrolysis and methylation analysis indicated that Gi-A1 was mainly composed of α-D-glucose, α-L-arabinose, and α-D-galactose with a molar ratio of 8.0:1.8:1.0. It can significantly stimulate spleen cell proliferation in vitro (P < 0.01). Published in Khimiya Prirodnykh Soedinenii, No. 1, pp. 13–14, January–February, 2009.     

Variable Volume Fed-Batch Fermentation for Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp<Emphasis Type="Italic">. lactis W28</Emphasis>

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l⁻¹) with 100 ml sucrose solution (190 g l⁻¹) being evenly fed (9–10 ml h⁻¹) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l⁻¹ in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l⁻¹, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.     

A New <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> Strain Producing Laccase. Use in Decolorization of Synthetics Dyes

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO₄ in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K _m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K _m = 700 μM), and pyrocatechol (K _m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions.     

Evaluation of a novel ELISA for serotonin: urinary serotonin as a potential biomarker for depression

Depression is a common disorder with physical and psychological manifestations often associated with low serotonin. Since noninvasive diagnostic tools for depression are sparse, we evaluated the clinical utility of a novel ELISA for the measurement of serotonin in urine from depressed subjects and from subjects under antidepressant therapy. We developed a competitive ELISA for direct measurement of serotonin in derivatized urine samples. Assay performance was evaluated and applied to clinical samples. The analytical range of the assay was from 6.7 to 425 μg serotonin/g creatinine (Cr). The limit of quantification was 4.7 μg/g Cr. The average recovery for spiked urine samples was 104.4%. Average intra-assay variation was 4.4%, and inter-assay variation was <20%. The serotonin analysis was very specific. No significant interferences were observed for 44 structurally and nonstructurally related urinary substances. Very good correlation was observed between urinary serotonin levels measured by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS; ELISA = 1.16 × LC-MS/MS − 53.8; r = 0.965; mean % bias = 11%; n = 18). Serotonin was stable in acidified urine for 30 days at room temperature and at −20 °C. The established reference range for serotonin was 54–366 μg/g Cr (n = 64). Serotonin levels detected in depressed patients (87.53 ± 4.89 μg/g Cr; n = 60) were significantly lower (p < 0.001) than in nondepressed subjects (153.38 ± 7.99 μg/g Cr). Urinary excretion of serotonin in depressed individuals significantly increased after antidepressant treatment by 5-hydroxy-tryptophane and/or selective serotonin re-uptake inhibitor (p < 0.01). The present ELISA provides a convenient and robust method for monitoring urinary serotonin. It is suitable to monitor serotonin imbalances and may be particularly helpful in evaluating antidepressant therapies.     

Isolation and Characterization of Exopolysaccharides Produced by the Cyanobacterium <Emphasis Type="Italic">Limnothrix redekei</Emphasis> PUPCCC 116

Limnothrix redekei PUPCCC 116, a filamentous cyanobacterium, has been identified through 16S rRNA gene sequencing. Exopolysaccharides (EPS) of this organism have been isolated and characterized chemically, and its rheological properties were compared with commercial xanthan. The organism produced 304 μg EPS/ml culture in 21 days. The rate of EPS production was maximum (313 μg EPS/mg protein/day) during the initial days of growth, and it decreased to 140 μg EPS/mg protein/day during 18-21 days of growth. Chemical analysis of EPS revealed the presence of glucose/mannose, ribose, rhamnose, and uronic acid. Fourier transformed infrared spectrum of EPS further revealed the presence of methyl and carboxyl groups besides C–N groups indicating the presence of peptidyl moieties. Elemental analysis of EPS showed the presence of 4.97% N. The organism under continuous light produced 102% more EPS compared to when grown under a light/dark cycle of 14/10 h. The rheological properties of EPS were comparable with commercial xanthan gum.     

Inhibition of Pro-inflammatory Secreted Phospholipase A2 by Extracts from <Emphasis Type="Italic">Cynara cardunculus L</Emphasis>.

The aim of the present work was to evaluate the anti-inflammatory properties of Cynara cardunculus L. (Asteraceae) during its growth using various solvents such as n-hexane, dichloromethane, acetone, and methanol for air-dried leaves and stems. The anti-inflammatory activities of crude extracts were evaluated by measuring the inhibition potency of mammalian non-pancreatic phospholipases A2 (hG-IIA). The methanol and acetone extracts of leaves harvested in February exhibit potent inhibition of hG-IIA (IC₅₀ = 50 and 70 μg/ml, respectively). However, the acetone extract of stems harvested in December inhibits the hG-IIA with a lower IC₅₀ around 130 μg/ml. Fractionation on silica gel and hydrophobic gel of the methanol extract of leaves harvested in February increases the inhibitory effect, and the IC₅₀ reached 10 μg/ml.     

Enhancement of Human γ-Interferon Production in Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Batch Cultivation

Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for γ-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of γ-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifnγ]. These conditions were considered as suitable conditions for the production of γ-interferon: 2.5× M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD₆₀₀ = 4.5). Under these conditions, dry cell weight of 6 ± 0.2 g/l and γ-interferon concentration of 2.15 ± 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of γ-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human γ-interferon. At improved conditions, a dry cell weight of 14 ± 0.2 g/l, concentration and overall productivity of γ-interferon 4.2 ± 0.1 g/l and 420 ± 10 mg/l h, respectively, were obtained.     

Contribution of Glycerol on Production of Poly(γ-Glutamic Acid) in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> NX-2

Glycerol would stimulate the production of poly(γ-glutamic acid) (γ-PGA) and decrease its molecular weight in Bacillus subtilis NX-2. When 20 g/l glycerol was added in the medium, the yield of γ-PGA increased from 26.7 ± 1.0 to 31.7 ± 1.3 g/l, and molecular weight of γ-PGA decreased from 2.43 ± 0.07 × 10⁶ to 1.86 ± 0.06 × 10⁶ Da. In addition, it was found that the decrease of γ-PGA chain length by glycerol would lead to the decrease of broth viscosity during the fermentation and enhanced the uptake of substrates, which could not only improve cell growth but also stimulate γ-PGA production. Moreover, it was also found that glycerol could effectively regulate molecular weight between 2.43 ± 0.07 × 10⁶ and 1.42 ± 0.05 × 10⁶ Da with the concentration ranging from 0 to 60 g/l. This was the first time to discover such contribution of glycerol on γ-PGA production in Bacillus genus. And the effects of glycerol on molecular weight of γ-PGA would be developed to be an approach for the regulation of microbial γ-PGA chain length, which is of practical importance for future commercial development of this polymer.     

Spectrophotometric Determination of Some Phenolic Antibiotics in Dosage Forms

 A simple and sensitive spectrophotometric method is described for the determination of some phenolic antibiotics namely: cefadroxil, amoxicillin and vancomycin. The method is based on the measurement of the orange yellow species produced when the drugs are coupled with diazotized benzocaine in triethylamine medium. The method is applicable over the range of 0.8–12 μg/ml for cefadroxil, 2–16 μg/ml for amoxicillin and 2–18 μg/ml for vancomycin. The formed compounds absorb at 455 nm for both cefadroxil and amoxicillin and at 442 nm for vancomycin. The proposed method has detection limits of 0.018 μg for cefadroxil, 0.0034 μg for amoxicillin and 0.0156 μg for vancomycin. The stoichiometric ratio for the studied compounds was found to be 1:1 and a proposal of the reaction pathway was made. The proposed method was applied for the analysis of the cited drugs in their pharmaceutical preparations. The results are in good agreement with those obtained by the official methods. Received February 7, 2000. Revision June 14, 2000.     

Ultra-fast two-dimensional microchip electrophoresis using SDS μ-CGE and microemulsion electrokinetic chromatography for protein separations

A poly(methyl methacrylate) microfluidic chip was used to perform a two-dimensional (2-D) separation of a complex protein mixture in short development times. The separation was performed by combining sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS μ-CGE) with microemulsion electrokinetic chromatography (μ-MEEKC), which were used for the first and second dimensions, respectively. Fluorescently labeled Escherichia coli cytosolic proteins were profiled by this 2-D approach with the results compared to a similar 2-D separation using SDS μ-CGE × μ-MEKC (micelle electrokinetic chromatography). The relatively short column lengths (effective length = 10 mm) for both dimensions were used to achieve separations requiring only 220 s of development time. High spot production rates (131 ± 11 spots min⁻¹) and reasonable peak capacities (481 ± 18) were generated despite the fact that short columns were used. In addition, the use of μ-MEEKC in the second dimension was found to produce higher peak capacities compared to μ-MEKC (481 ± 18 for μ-MEEKC and 332 ± 17 for μ-MEKC) due to the higher plate numbers associated with μ-MEEKC.     

Sequential Injection Kinetic Flow Assay for Monitoring Glycerol in a Sugar Fermentation Process by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A sequential injection system to monitor glycerol in a Saccharomyces cerevisiae fermentation process was developed. The method relies on the rate of formation of nicotinamide adenine dinucleotide in its reduced form (NADH, measured spectrophotometrically at 340 nm) from the reaction of glycerol with NAD⁺ cofactor, catalysed by the enzyme glycerol dehydrogenase present in solution. This procedure enables the determination of glycerol between 0.046 and 0.46 g/l, (corresponding to yeast fermentation samples with concentrations up to 50 g/l) with good repeatability (relative standard deviation for n = 10 lower than 2.2% for three different samples) at a sampling frequency of 25/h. The detection and quantification limits using a miniaturised spectrophotometer were 0.13 and 0.44 mM, respectively. Reagent consumption was of 0.45 μmol NAD⁺ and 1.8 μg enzyme per assay, and the waste production was 2.8 ml per determination. Results obtained for samples were in agreement with those obtained with a high-performance liquid chromatography method.     

Certification of an animal feed reference material for the organochlorine pesticide contents: BCR CRM 115

 An animal feed reference material has been prepared and enriched with 15 organochlorine pesticides at levels close to the maximum residue limits set up in the European Directive 74/63/EEC. The homogeneity of the material has been assessed on the basis of the pesticide contents at a level of sample intake of 5 g. The one year stability study revealed an instability of heptachlor, o,p′-DDT, p,p′-DDT, α-HCH and γ-HCH (decrease in content) and p,p′-TDE (increase) at +37 °C; at +20 °C no instability could be demonstrated. The material has been certified in an interlaboratory certification study for: HCB (0.0194 mg/kg), β-HCH (0.023 mg/kg), γ-HCH (0.0218 mg/kg), heptachlor (0.019 mg/kg), γ-Chlordane (0.048 mg/kg), α-Endosulfan (0.046 mg/kg), Dieldrin (0.018 mg/kg), Endrin (0.046 mg/kg), o,p′-DDT (0.046 mg/kg) and p,p′-DDE (0.047 mg/kg). The α-HCH isomer, Aldrin, β-Heptachloro-epoxide, p,p′-DDT and p,p′-TDE could not be certified. The methods used for the certification are described as well as the major difficulties encountered in the study. Received: 1 July 1996/Revised: 14 October 1996/Accepted: 16 October 1996     

Preconditioning of Axillary Buds in Thidiazuron-Supplemented Liquid Media Improves In Vitro Shoot Multiplication in <Emphasis Type="Italic">Nyctanthes arbor-tristis</Emphasis> L.

An efficient tissue culture technology has been designed for mass multiplication of Nyctanthes arbor-tristis L. by preculturing nodal explants in thidiazuron (TDZ)-supplemented liquid Murashige and Skoog (MS) media. Direct inoculation of nodal segments on semi-solid MS medium augmented with various concentrations of TDZ (0.1 to 0.9 μM) produced shoots but with low regeneration response and few shoots per explant. Hence, nodal explants were pretreated with greater concentrations of TDZ (5 to 100 μM) in liquid MS media for different durations (4, 8, 12, and 16 days) with the aim of improving shoot regeneration response from cultured explants. After pretreatment, explants were transferred to agar-solidified hormone-free MS medium. Best response in terms of percent regeneration (94%), number of shoots per explant (20.00 ± 1.15), and greatest shoot length (7.23 ± 0.83 cm) were obtained with nodal segments pretreated in75 μM TDZ for 8 days. Similarly, root induction was obtained from pulse-treated microshoots for 24 h with 200 μM indole-3-butyric acid (IBA) followed by their transfer to 1/2 MS medium which produced an average of 5.50 ± 0.92 roots per microshoot. The rooted plantlets were transplanted to soil with 80% success rate.