Isolation, characterization, and multimodal evaluation of novel glycolipid biosurfactant derived from Bacillus species: A promising Staphylococcus aureus tyrosyl-tRNA synthetase inhibitor through molecular docking and MD simulations.

Glycolipid-based biosurfactants (BSs), known for their intriguing and diverse properties, represent a largely uncharted territory in the realm of potential biomedical applications. This field holds great promise yet remains largely unexplored. This investigation provides new insights into the isolation, characterization, and comprehensive biomedical assessment of a novel glycolipid biosurfactant derived from Bacillus species, meeting the growing demand for understanding its multifaceted impact on various biomedical issues. Within this framework, two glycolipids, BG2A and BG2B, emerged as the most proficient strains in biosurfactant (BS) production. The biosurfactants (BSs) ascertained as glycolipids via thin layer chromatography (TLC) exhibited antimicrobial activity against S. aureus and E. coli. Both isolates exhibited anticancer effects against cervical carcinoma cells and demonstrated significant anti-biofilm activity against V. cholerae. Moreover, molecular docking and molecular dynamics (MD) simulations were employed to explore their antimicrobial resistance properties against Tyrosyl-tRNA synthetase (TyrRS) of Staphylococcus aureus, a well-annotated molecular target. Characterization and interpretation using Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1H and 13C NMR) confirmed that the BSs produced by each strain were glycolipids. These findings suggest that the isolated BSs can serve as effective agents with antibiofilm, antimicrobial, antioxidant, and anticancer properties, in addition to their considerable antibacterial resistance attributes.

Improved Chrysin Production by a Combination of Fermentation Factors and Elicitation from Chaetomium globosum.

Flavonoids encompass a heterogeneous group of secondary metabolites with exceptional health benefits. Chrysin, a natural dihydroxyflavone, possesses numerous bioactive properties, such as anticancer, antioxidative, antidiabetic, anti-inflammatory, etc. However, using traditional sources of chrysin involves extracting honey from plants, which is non-scalable, unsustainable, and depends on several factors, including geography, climatic conditions, and the season, which limits its production at a larger scale. Recently, microbial production of desirable metabolites has garnered attention due to the cost-effectiveness, easy scale-up, sustainability, and low emission of waste. We previously reported for the first time the chrysin-producing marine endophytic fungus Chaetomium globosum, associated with a marine green alga. To extend our understanding of chrysin biosynthesis in C. globosum, in the present study, we have assessed the presence of flavonoid pathway intermediates in C. globosum extracts using LC-MS/MS. The presence of several key metabolites, such as dihydrokaempferol, chalcone, galangin, baicalein, chrysin, p-Coumaroyl-CoA, and p-Cinnamoyl-CoA, indicates the role of flavonoid biosynthesis machinery in the marine fungus. Further, we have aimed to enhance the production of chrysin with three different strategies: (1) optimizing the fermentation parameters, namely, growth medium, incubation time, pH, and temperature; (2) feeding key flavonoid pathway intermediates, i.e., phenylalanine and cinnamic acid; (3) elicitation with biotic elicitors, such as polysaccharide, yeast extract, and abiotic elicitors that include UV radiation, salinity, and metal stress. The combined effect of the optimized parameters resulted in a 97-fold increase in the chrysin yield, resulting in a fungal cell factory. This work reports the first approach for enhanced production of chrysin and can serve as a template for flavonoid production enhancement using marine endophytic fungi.

Inhibition of Autophagy Increases Cell Death in HeLa Cells through Usnic Acid Isolated from Lichens.

The Western Ghats, India, is a hotspot for lichen diversity. However, the pharmacological importance of lichen-associated metabolites remains untapped. This study aimed to evaluate the cytotoxic potential of lichens of this region. For this, sixteen macrolichens were collected and identified from two locations in the Western Ghats. The acetone extract of Usnea cornuta (UC2A) showed significant cytotoxicity towards multiple human cancer cell lines. Interestingly, co-treatment with chloroquine (CQ), an autophagy inhibitor, increased the cytotoxic potential of the UC2A extract. A gas chromatography mass spectrometry (GCMS) study revealed usnic acid (UA), atraric acid and barbatic acid as the dominant cytotoxic compounds in the UC2A extract. Further, UA was purified and identified from the UC2A extract and evaluated for cytotoxicity in HeLa cells. The monodansyl cadaverine and mitotracker red double staining revealed the autophagy-inducing activities of UA, and the inhibition of autophagy was confirmed via CQ treatment. Autophagy inhibition increased the cytotoxicity of UA by 12-16% in a concentration-dependent manner. It also increased lipid peroxidation, ROS levels and mitochondrial depolarization and decreased glutathione availability. A decrease in zeta potential and a 40% increase in caspase 3/7 activity were also noted after CQ treatment of UA-treated cells. Thus, cytotoxicity of UA can be increased by inhibiting autophagy.

Infrared spectroscopy and flow cytometry studies on the apoptotic effect of nano-chrysin in HeLa cells.

Mapping the structural changes in membrane lipids, proteins, polysaccharides and nucleic acids has opened new channels for understanding the mode of action of anticancer natural products. Earlier, we synthesized chrysin nanoparticles (NChr) with good bioavailability, and characterized its size, surface charge, entrapment efficiency, and drug release pattern using PLGA polymer. NChr induced concentration dependent cytotoxicity in HeLa cells with an IC50 of 61.54 ± 1.2 µM in comparison with free chrysin with IC50 of 86.51 ± 2.9 µM. Since nanoparticles interact dynamically with cell membranes, organelles, proteins and DNA, it is necessary to understand the interplay of nanodrug induced macromolecular changes in cancer cells. In this work, we obtained signatures of NChr-induced biochemical changes in HeLa cells by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy technique coupled with flow cytometry. NChr induced cell membrane disruption, G1 phase cell cycle arrest, and increased externalization of phosphatidylserine leading to apoptosis indicating the biochemical perturbations in membrane lipids and DNA of HeLa cells in comparison with untreated cells. The 1300-1000 cm-1 spectral region indicated NChr interaction with the ribose sugar backbone and DNA denaturation. Spectral range 1800-1400 cm-1 indicated a concentration dependent decrease in α helical and ß sheet structures which may lead to protein degradation during apoptosis. The spectral range 3000-2800 cm-1 indicated the lipid peroxidation in response to NChr treatment. This is the first study describing the bio-macromolecular changes induced by a nano encapsulated drug and can pave the way to investigate unconventional modes of action for bioactive formulations.

Usnic acid induced changes in biomolecules and their association with apoptosis in squamous carcinoma (A-431) cells: A flow cytometry, FTIR and DLS spectroscopic study.

Many natural products induce apoptotic cell death in cancer cells, though studies on their interactions with macromolecules are limited. For the first time, this study demonstrated the cytotoxic potential of usnic acid (UA) against squamous carcinoma (A-431) cells and the associated changes in cell surface proteins, lipids and DNA by attenuated total reflection- fourier transform infrared spectroscopy (ATR-FTIR) and dynamic light scattering (DLS) spectroscopic studies. The IC50 for UA was 98.9 µM after treatment of A-431 cells for 48 h, while the IC50 reduced to 39.2 µM after 72 h of incubation time. UA induced oxidative stress in treated cells as confirmed by DCFHDA flow cytometry assay, depletion in reduced glutathione and increase in lipid peroxidation. The oxidative stress resulted in conformation change in amide I, amide II protein bands and DNA as observed by ATR-FTIR in UA treated A-431 cells. Shift in secondary structures of proteins from α helix to ß sheets and structural changes in DNA was observed in UA treated A-431 cells. An increase in the band intensity of phospholipids, increased distribution of lipid and change in membrane potential was noted in UA treated cells, which was confirmed by externalization of phosphatidylserine to the outer membrane by annexin V-FITC/PI assay. Increase in mitochondrial membrane potential, cell cycle arrest at G0/G1 phase by flow cytometry and activation of caspase-3/7 dependent proteins confirmed the UA induced apoptosis in treated A-431 cells. FTIR and DLS spectroscopy confirmed the changes in biomolecules after UA treatment, which were associated with apoptosis, as observed by flow cytometry.

Multi-functional bioactive secondary metabolites derived from endophytic fungi of marine algal origin.

Endophytic fungi from marine macroalgae are endowed with various pharmacologically active metabolites. This study mined, identified and screened endophytic fungi (EF) isolated from marine algae from the west coast of South India for screening anticancer, antioxidant and antimicrobial secondary metabolite producers. Five EF isolated from sampled marine algae were identified using morphology and ITS based identification as Grammothele fuligo, Rigidoporus vinctus, Cystobasidium minutum, Candida railenensis and Pichia kudriavzevii. After culturing 20 days on PDA medium, the ethyl acetate extracts of Cystobasidium minutum showed potent antimicrobial activity against P. aeruginosa with an IC50 value of 458.7 ± 1.021 µg/mL whereas Pichia kudriavzevii extracts showed promising antioxidant activity (IC50 value of 65.78 ± 1.082 µg/mL, 38.74 ± 1.040 µg/mL and 32.01 ± 1.018 µg/mL for DPPH assay, ABTS assay and FRAP assay respectively) and high cytotoxic activity against MG63 cell line (IC50 = 145.1 ± 1.086 µg/mL, no activity against U87 cells). The phytochemical screening of the extracts unveiled the existence of diverse groups of secondary metabolites. Further, Gas Chromatography Mass Spectroscopy (GC-MS) analysis of the extract revealed the presence of compounds that are known to be antibacterial, antioxidant and cytotoxic. These results indicate that marine derived endophytes could be potent sources for multi-functional bioactive compounds and may find prospective application in pharmaceutical industry.

Polyphenol oxidase (PPO) arm of catecholamine pathway catalyzes the conversion of L-tyrosine to L-DOPA in Mucuna pruriens (L.) DC var. pruriens: An integrated pathway analysis using field grown and in vitro callus cultures.

Mucuna pruriens (L.) DC var. pruriens is the natural source for L-DOPA, precursor of the neurotransmitter dopamine, used widely in the treatment of Parkinson's disease. However, L-DOPA synthesis in plants is mediated either by Catecholamine (CA) pathway or alternate pathway catalyzed by Cytochrome P450 (CYP450) class of enzymes. Interestingly, the CA pathway itself can be initiated either by tyrosine hydroxylase (TH) or polyphenol oxidase (PPO). The CA pathway mediated synthesis of L-DOPA has not yet been proved in M. pruriens albeit strong indications. Therefore, the present investigation is focused on metabolite analysis of major intermediates of CA pathway up to the formation of dopamine and expression analysis of the selected genes, in different tissues and callus cultures. The four major intermediates, L-tyrosine, tyramine, L-DOPA and dopamine, were detected using NMR spectroscopy and quantified by HPLC in the callus cultures and in different tissues of the field plant, respectively. The various stages of leaf tissue were also analyzed for metabolite profiling. The relative amount of intermediates detected during the ontogeny of leaf indicates that PPO mediated conversion of L-tyrosine to dopamine through L-DOPA is relatively higher compared to dopamine production from tyramine. Among the two possible enzymes, activity of PPO was 6.5-fold more than TH in metabolically active young leaves compared to intermediate leaves. The gene expression profiles comprising upstream genes of L-tyrosine synthesis and downstream up to dopamine synthesis shows strong correlation with L-DOPA synthesis. The study validates CA pathway mediated synthesis of L-DOPA with PPO as candidate enzyme, in M. pruriens.

Optimization of PhysicoChemical Parameters for Production of Cytotoxic Secondary Metabolites and Apoptosis Induction Activities in the Culture Extract of a Marine Algal-Derived Endophytic Fungus Aspergillus sp.

The endophytic fungal community in the marine ecosystem has been demonstrated to be relevant source of novel and pharmacologically active secondary metabolites. The current study focused on the evaluation of cytotoxic and apoptosis induction potential in the culture extracts of endophytic fungi associated with Sargassum muticum, a marine brown alga. The cytotoxicity of the four marine endophytes, Aspergillus sp., Nigrospora sphaerica, Talaromyces purpureogenus, and Talaromyces stipitatus, was evaluated by the MTT assay on HeLa cells. Further, several physicochemical parameters, including growth curve, culture media, and organic solvents, were optimized for enhanced cytotoxic activity of the selected extract. The Aspergillus sp. ethyl acetate extract (ASE) showed maximum cytotoxicity on multiple cancer cell lines. Chemical investigation of the metabolites by gas chromatography-mass spectroscopy (GC-MS) showed the presence of several compounds, including quinoline, indole, 2,4-bis(1,1-dimethylethyl) phenol, and hexadecenoic acid, known to be cytotoxic in ASE. The ASE was then tested for cytotoxicity in vitro on a panel of six human cancer cell lines, namely, HeLa (cervical adenocarcinoma), MCF-7 (breast adenocarcinoma), Hep G2 (hepatocellular carcinoma), A-549 (lung carcinoma), A-431 (skin/epidermis carcinoma), and LN-229 (glioblastoma). HeLa cells were most vulnerable to ASE treatment with an IC50 value of 24 ± 2 µg/ml. The mechanism of cytotoxicity exhibited by the ASE was further investigated on Hela cells. The results showed that the ASE was capable of inducing apoptosis in HeLa cells through production of reactive oxygen species, depolarization of mitochondrial membrane, and activation of the caspase-3 pathway, which shows a possible activation of the intrinsic apoptosis pathway. It also arrested the HeLa cells at the G2/M phase of the cell cycle, eventually leading to apoptosis. Through this study, we add to the knowledge about the marine algae associated with fungal endophytes and report its potential for purifying specific compounds responsible for cytotoxicity.

Endophytic fungus, Chaetomium globosum, associated with marine green alga, a new source of Chrysin.

The marine ecosystem is an extraordinary reserve of pharmaceutically important, bioactive compounds even in this "synthetic age". Marine algae-associated endophytic fungi have gained prominence as an important source of bioactive compounds. This study was conducted on secondary metabolites of Chaetomium globosum-associated with marine green alga Chaetomorpha media from the Konkan coastline, India. Its ethyl acetate extract (CGEE) exhibited an IC50 value of 7.9 ± 0.1 µg/mL on MCF-7 cells. CGEE exhibited G2M phase cell cycle arrest, ROS production and MMP loss in MCF-7 cells. The myco-components in CGEE contributing to the cytotoxicity were found by Gas Chromatography/Mass Spectrometry analyses. Chrysin, a dihydroxyflavone was one of the forty-six myco-components which is commonly found in honey, propolis and passionflower extracts. The compound was isolated and characterized as fungal chrysin using HPLC, UV-Vis spectroscopy, LC-MS, IR and NMR analyses by comparing with standard chrysin. The purified compound exhibited an IC50 value of 49.0 ± 0.6 µM while that of standard chrysin was 48.5 ± 1.6 µM in MCF-7 cells. It induced apoptosis, G1 phase cell cycle arrest, MMP loss, and ROS production. This is the first report of chrysin from an alternative source with opportunities for yield enhancement.

L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers.

Mucuna pruriens L., commonly known as velvetbean or cow-itch, is a self-pollinated tropical legume of the family Fabaceae, known for its medicinal properties. The active principle L-DOPA extracted from the plant is a potent drug used in the treatment of Parkinson's disease. Although, it is hypothesized that a single step reaction can produce L-DOPA, the presence of optional routes makes the pathway more intricate. For instance, the catecholamine biosynthetic pathway, which leads to L-DOPA production, could occur by hydroxylation of tyrosine to L-DOPA either by polyphenol oxidase (PPO) or tyrosine hydroxylase (TH). Furthermore, Cytochrome P450 (CYP) enzymes can also cause hydroxylation of tyrosine, resulting in L-DOPA synthesis. Therefore, the present investigation was focused on validating the step, which catalyzes the synthesis of L-DOPA, at the biochemical and molecular levels. Enzyme inhibitor studies showed significant inhibition of PPO enzyme with corresponding decrease in L-DOPA synthesis while TH and CYP inhibition had no effect on L-DOPA synthesis. Activity staining of non-denaturing PAGE gel for PPO and TH showed activity only to PPO enzyme. Following in-gel assay and tryptic digestion of the excised stained gel portion, peptide recovery and LC-MS/MS analysis were performed. Degenerate primers based on peptide sequence resulted in an 800bp amplicon. The subsequent sub-cloning, RACE analysis and BLAST search resulted in the isolation of full-length PPO coding sequence of 1800 bp. Structure prediction and phylogenetic analysis of the obtained sequence revealed strong similarity to other plant PPO's like Glycine max, Vigna radiata and Vicia faba of the same family.

Antiproliferative and Antioxidative Bioactive Compounds in Extracts of Marine-Derived Endophytic Fungus Talaromyces purpureogenus.

Endophytic fungi are now recognized as sources of pharmacologically beneficial, novel bioactive compounds. This study was carried out to evaluate antiproliferative and antioxidative potential of a seaweed endophytic fungus Talaromyces purpureogenus. Extracts with different solvents of the fungus grown on different liquid media were assayed for the antiproliferative and antioxidative activities. Tested 6 cancer cell lines, the highest antiproliferative activity was observed in ethyl acetate extract of total culture grown in Potato Dextrose Broth for 28 days in a dose-dependent manner. The highest antioxidative activity was observed in hexane extract of fungal culture grown in Malt Extract Broth for 21 days. Analyzed for secondary metabolites, the extract revealed the presence of phenolics, alkaloids, flavonoids, steroids and terpenoids. Further, Gas Chromatography Mass Spectroscopy (GCMS) analysis of the extract revealed the presence of several compounds including 3-nitropropanoic acid, 4H-pyran-4-one 5-hydroxy-2-(hydroxymethyl), hexadecanoic acid, and octadecanoic acid, known to be cytotoxic or antioxidative. Among different cell lines tested, HeLa cells were the most vulnerable to the treatment of the fungal extract with an IC50 value of 101 ± 1 µg/mL. The extract showed no significant cytotoxicity to the normal human embryonic kidney cell line (HEK 293 T) in the MTT assay. The ethyl acetate extract induced membrane damage and mitochondrial depolarization and thereby apoptosis and cytotoxicity in HeLa cells. The study marks marine-derived endophytes as potential sources for discovery of novel drugs.

Isolation, Purification and Characterization of a Novel Steroidal Saponin Cholestanol Glucoside from Lasiodiplodia theobromae that Induces Apoptosis in A549 Cells.

Search for novel anticancer lead molecules continues to be a major focus of cancer research due to the limitations of existing drugs such as lack of tumor selectivity, narrow therapeutic index and multidrug resistance of cancer types. Natural molecules often possess better pharmacokinetic traits compared to synthetic molecules as they continually evolve by natural selection process to interact with biological macromolecules. Microbial metabolites constitute nearly half of the pharmaceuticals in market today. Endophytic fungi, owing to its rich chemical diversity, are viewed as attractive sources of novel bioactive compounds. In the present study, we report the purification and characterization of a novel steroidal saponin, cholestanol glucoside (CG) from Saraca asoca endophytic fungus Lasiodiplodia theobromae. The compound was assessed for its cytotoxic potentialities in six human cancer cell lines, A549, PC3, HepG2, U251, MCF7 and OVCAR3. CG exhibited significant cytotoxicities towards A549, PC3 and HepG2 among which A549 cells were most vulnerable to CG treatment. However, CG treatment exhibited negligible cytotoxicity in non malignant human lung fibroblast cell line (WI-38). Induction of cell death by CG treatment in A549 cells was further investigated. CG induced the generation of reactive oxygen species (ROS) and mitochondrial membrane permeability loss followed by apoptotic cell death. Mitochondrial membrane depolarization and apoptotic cell death in CG treated A549 cells were completely blocked in presence of an antioxidant, N-acetyl cysteine (NAC). Hence it could be concluded that CG initiates apoptosis in cancer cells by augmenting the basal oxidative stress and that the generation of intracellular ROS is crucial for the induction of apoptosis.

Cucumber chloroplast trnL(CAA) gene: nucleotide sequence and in vivo expression analysis in etiolated cucumber seedlings treated with benzyladenine and light.

The nucleotide sequence of a 714 bp BamHI-EcoRI fragment of cucumber chloroplast DNA was determined. The fragment contained a gene for tRNA(Leu) together with its flanking regions. The trnL(CAA) gene sequence is about 99% in similarity to broad bean, cauliflower, maize, spinach and tobacco corresponding genes. The relative expression level of the gene was determined by Northern (tRNA) gel blot and Northern (total cellular RNA) slot-blot analyses using the trnL gene probe in 6-day old etiolated cucumber seedlings and the seedlings that had been kept in the dark (dark-grown), treated with benzyladenine (BA) and kept in the dark (BA-treated dark-grown), illuminated (light-grown), and treated with BA and illuminated (BA-treated light-grown), for additional 4, 8 or 12 hr. The trnL transcripts and tRNA(Leu) levels in BA-treated dark-grown seedlings were 5 and 3 times higher, respectively after 4 hr BA treatment, while in the BA treated light-grown seedlings the level of trnL transcripts was only 3 times higher and had no detectable effect on mature tRNA(Leu) when compared to the time-4 hr dark-grown seedlings. However, the level of mature tRNA(Leu) did not show marked changes in the light-grown seedlings, whereas the level of trnL transcripts increases 3 times after 8 hr illumination of dark-grown seedlings. These data indicate that both light and cytokinin can signal changes in plastid tRNA gene expression. The possible regulatory mechanisms for such changes are discussed.

Light- and cytokinin-induced changes in the levels of leucine and tyrosine isoaccepting tRNA species and modified nucleotide contents of total tRNA in cucumber seedlings.

We have determined relative levels of chloroplast leucine and tyrosine isoaccepting tRNAs and modified nucleotide contents from total tRNAs isolated from dark-grown, light-grown, N6-isopentenyladenine (i6A)-treated dark-grown and i6A-treated light-grown cucumber seedlings. Significant increases in the relative amounts of tRNA(Leu)2 and tRNA(Leu)3 were observed in the i6A-treated dark-grown seedlings compared to dark-grown, light-grown and i6A-treated light-grown seedlings. On the other hand, i6A-treated light-grown seedlings tRNA(Tyr)1 increased to 85% of total tRNAs(Tyr) from about 9% in light-grown seedlings and tRNA(Tyr)2 decreased to 15% compared with 91% in light-grown seedlings. Analysis of modified nucleotide of total tRNAs indicated that pT, pI, pm1A, pm5C, pGm, pm1G, pm2G and pm7G contents were significantly higher in the total tRNA of i6A-treated dark-grown seedlings than those from untreated dark-grown seedlings. Illumination of 8-day-old dark-grown seedlings for 12 h increased the contents of pT, pI, pGm and pm1G when compared to 8-day-old dark-grown seedlings with extended growth for 12 h in dark. On the contrary, i6A had no stimulatory effect in the contents of modified nucleotide in the light-grown seedlings.

Purification of antibodies for the cytokinin N6-(delta 2-isopentenyl) adenosine by affinity chromatography.

Isopentenyl adenosine antibodies useful in the investigations of the "cytokinin" functions of isopentenyl adenosine were purified by affinity chromatography. Using different affinity columns, the antibodies were purified to near complete purity. Analyses of the purified proteins revealed the presence of isopentenyl adenosine binding proteins in normal rabbit serum, which presence supports a suggested role for isopentenyl adenosine and its related compounds in animal cell division in vivo.

Immunospecific binding of tRNA precursors containing N6-(delta 2-isopentenyl) adenosine.

Antibodies specific for N6-(delta 2-isopentenyl) adenosine (i6A) were immobilized on Sepharose and this adsorbent (Sepharose-anti-i6A) was used to selectively isolate bacteriophage T4 tRNA precursors containing i6A/ms2i6A from an unfractionated population of 32P-labeled T4 RNAs. The results showed that antibodies to i6A selectively bound only those tRNA precursors containing i6A/ms2i6A. Binding of tRNA precursors by antibody and specificity of the binding was assessed by membrane binding using 32P-labeled tRNA precursor. Binding was highly specific for i6A/ms2i6A residues in the tRNA precursors. This binding can be used to separate modified from unmodified precursor RNAs and to study the biosynthetic pathways of tRNA precursors.

Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis sativus L. var. Guntur Seedlings.

N(6)-(Delta(2)-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N(6)-(Delta(2)-isopentenyl) adenosine antibodies retained tRNAs containing N(6)-(Delta(2)-isopentenyl) adenosine and N(6)-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N(6)-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N(6)-(Delta(2)-isopentenyl) adenosine, and N(6)-(Delta(2)-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N(6)-(Delta(2)-isopentenyl) adenosine and N(6)-(4-hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNA(Phe) and tRNA(Tyr) contained cytokinins whereas tRNA(Ala) did not.