The KMT2A recombinome of acute leukemias in 2023.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

The MLL recombinome of acute leukemias in 2017.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

The MLL recombinome of acute leukemias in 2013.

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.

Differential regulation and distribution of acridone synthase in Ruta graveolens.

Cell suspension cultures of Ruta graveolens L. accumulate polyketide metabolites such as acridone alkaloids and flavonoid pigments. Whereas flavonoid synthesis is induced by light, the production of alkaloids can be enhanced in dark-cultured cells by treatment with fungal elicitors. Acridone synthase (ACS) catalyzes the committed condensing reaction of acridone biosynthesis yielding 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl- and malonyl-CoAs. The reaction proceeds in a manner analogous to that of chalcone synthase (CHS) which catalyzes the first committed step in flavonoid biosynthesis and cDNA and protein sequences of Ruta ACS possess a high degree of sequence homology to heterologous CHSs. ACS transcript abundance and specific activity were monitored in cultured R. graveolens cells irradiated either continuously with white light or treated with fungal elicitor over a period of 24 h and found to increase transiently upon elicitor treatment and to decrease upon light irradiation. Immunodetection with a rabbit polyclonal ACS antiserum revealed that the amounts of ACS polypeptide decreased slightly in light-irradiated cells but increased in elicitor-treated Ruta cells. Fluorescence microscopy and tissue print hybridizations were employed to aid in localizing the sites of storage and biosynthesis of acridone alkaloids in Ruta plants. Yellow fluorescing alkaloids were detected particularly in root tissue adjacent to the rhizodermis, but also in the endodermis and vascular tissue of the hypocotyl. ACS transcript abundance in situ followed the same spatial pattern, indicating that the synthesis of acridones likely proceeds at all sites of deposition rather than exclusively in the root. Expression in planta and the induction response of ACS suggest that the alkaloids serve as phytoanticipins or phytoalexins in the defense of Ruta particularly to soil-borne pathogens or as feeding deterrents.

Molecular cloning and heterologous expression of acridone synthase from elicited Ruta graveolens L. cell suspension cultures.

Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in lambda 2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibit high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.

Acridone Alkaloids from Cell Suspension Cultures of Thamnosma montana.

Cell Suspension cultures of THAMNOSMA MONTANA were grown in Gamborg B5 medium which was best suited for acridone alkaloid formation. From the lyophilized cell material nine acridones and three acridone-monoglucosides have been isolated which were found for the first time in the genus THAMNOSMA. Moreover, N-methylacridone which is known from the intact plant was found. Gravacridonol monoglucoside was identified as a new alkaloid. Interestingly, most isolated alkaloids belong to the dihydrofuroacridones.

Purification and properties of acridone synthase from cell suspension cultures of Ruta graveolens L.

Acridone synthase has been purified from cell suspension cultures of Ruta graveolens using a combination of gel filtration and ion exchange chromatography. The purified enzyme has an apparent molecular weight of 69 kDa on gel filtration and a subunit structure on SDS-PAGE of 40 kDa. The apparent Km-values are 10.64 microM and 32.8 microM for N-methylanthraniloyl-CoA and malonyl-CoA, respectively. Tryptic digestion of the homogeneous acridone synthase was performed. Seven of the peptides were chosen for microsequencing. The homology of the amino acid sequences from this particular polypeptide and corresponding peptides from chalcone synthase 3 from garden pea amounted to 76%.

Steric course of the N-methylation in the biosynthesis of ergot alkaloids by Claviceps purpurea.

Using the chiral methyl group methodology, the methylation step in the biosynthesis of ergot alkaloids catalyzed by the enzyme AdoMet:dimethylallyltryptophan N-methyltransferase was found to proceed with net inversion of methyl group configuration. The enzyme thus conforms to the majority of methyltransferases studied which mediate a direct SN2 transfer of the methyl group from AdoMet to the acceptor nucleophile in a ternary enzyme substrate complex.

[Synthesis and pharmacologic properties of pranolium and its optical isomers]. / Synthese und pharmakologische Eigenschaften von Pranolium und seinen optischen Isomeren.

Propranolol is a well-known powerful betareceptor-blocking agent. Its quaternary dimethyl derivative, designated as pranolium was firstly prepared by Lucchesi. Compared to propranolol it possesses no betareceptor-blocking activity and no local anaesthetic properties but shows the same antiarrhythmic action as the starting material. The synthesis of pranolium and its optical isomers starting from the corresponding propranolol derivatives is described. Their pharmacological activities have been tested. No significant differences regarding the pharmacological action could be observed.

Biotransformation of a [14CH3]-2-Methylaminobenzophenone by Plant Cell Cultures.

2-[ (14)C]-Methylamino-2',4'-dimethoxy-6'-hydroxybenzophenone ( 4) was synthesized and administered to RUTA GRAVEOLENS cell suspension cultures. Compound 4 was not incorporated into acridone alkaloids but glucosylated giving 7. This reaction takes place also in cell suspension cultures of ADHATODA VASICA and PEGANUM HARMALA.

Biosynthesis of phenylalanine and tyrosine in claviceps.

Some enzymes of the terminal steps of phenylalanine and tyrosine biosyntheses were investigated in various alkaloid-forming ergot strains. All strains studied utilize both arogenate and phenylpyruvate as an intermediate in L-phenylalanine synthesis. L-Tyrosine is preferentially or exclusively synthesized via the arogenate pathway. No feedback inhibition of arogenate pathway enzymes by aromatic amino acids was observed.

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra.

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.

Influence of tryptophan and related compounds on ergot alkaloid formation in Claviceps purpurea (FR.) Tul.

L-Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine-accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high-yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mM stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5-Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

N-Methylation of Anthranilic Acid to N-Methylanthranilic Acid by Cell-free Extracts of Ruta graveolens Tissue Cultures.

Cell-free extracts from acridone synthesizing cell suspension cultures of RUTA GRAVEOLENS L. catalyze the N-methylation of anthranilic acid using S-adenosyl-L-methionine as methyl donor. The stability of enzyme preparations was remarkably high during storage at -20 degrees C. Optimum activity was exhibited at pH 8.2, Mg (2+) was not required for maximum activity and EDTA did not affect the reaction rate. The rate of N-methylanthranilic acid formation was shown to be linear for about 45 min and was proportional to the protein concentration up to at least 0.350 mg of the enzyme preparation. In a number of suspension cultures of plant species not belonging to the Rutaceae this particular N-methyltransferase was not found. Apparantly N-methylation of anthranilic acid is the first pathway-specific reaction in acridone alkaloid biosynthesis.

Biosynthesis of rutacridone: the N-methylation step.

The N-methylation of rutacridone was investigated. This dihydrofuroacridone derivative ist the main alkaloid in cell suspension cultures of RUTA GRAVEOLENS, strain R-20. The N-methyl group is provided by L-methionine. N-methylanthranilic acid serves as excellent precursor of rutacridone. Furthermore this particular precursor could be trapped after feeding anthranilic acid in short term experiments.

Indole Alkaloid Formation and Storage in Cell Suspension Cultures of Catharanthus roseus.

The localization and storage of alkaloids were investigated in a low producing cell suspension culture of CATHARANTHUS ROSEUS(L.) G. Don. (Apocynaceae). Fluorescence microscopy and electron microscopy indicate alkaloid accumulation to occur inside the vacuoles of particular cells. These alkaloid storage cells exhibit a vacuolar pH of 3, while "normal" cells of a suspension culture have a vacuolar pH of about 5. Alkaloids are taken up in their unprotonated forms, trapped by protonation inside the vacuole and are accumulated there. The differentiation of alkaloid storage cells depends both on the cell line and the growth conditions and seems to be a prerequisite for the accumulation of alkaloids in cell suspension cultures of CATHARANTHUS ROSEUS.

Studies on tissue cultures of the genus Cinchona L. : In vitro mass propagation through meristem-derived plants.

A procedure for the in vitro clonal mass propagation of shoots and plants of two Cinchona species derived from single shoot tips is described. Special attention is given to rooting problems of the shoots under in vitro conditions.

Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures.

Initiation and culture of callus and cell suspensions of Cinchona ledgeriana and C. succirubra as well as the successful isolation and selection of a high-yielding alkaloid-forming strain derived from the leaf rachis of a C. succirubra plant are described. Results of feeding experiments with L-tryptophan using two different culture procedures are presented and discussed. Maximum alkaloid yields of up to 0.9% (based on dry weight) or 6.35 mg/l have been obtained.

A new strain of Claviceps purpurea accumulating tetracyclic clavine alkaloids.

A new strain of Claviceps was isolated from a blokked mutant of Claviceps purpurea. This strain accumulates substantial amounts of clavine alkaloids (2 g/l). The alkaloid fraction is composed of chanoclavine-I ( approximately 10%) and a mixture of agroclavine/elymoclavine (90%). Most suitable for alkaloid production in submerged culture is an ammoncitrate/sucrose medium. The genealogy of the new strain, designated Pepty 695/ch-I is the following one: Pepty 695/S (ergotoxine producer) --> Pepty 695/ch (secoergoline producer) --> Pepty 695/ch-I (tetracyclic clavine producer).