Brassinosteroids: Methods and Protocols


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Original Research ARTICLE

In the s, it was discovered in Arabidopsis that BRs are essential plant hormones through analysis of mutant plants unable to naturally synthesize BRs. These Arabidopsis mutants which show characteristic dwarfism, e. Science , ; dim: Takahashi et al. Genes Dev. Plant J. Cell, 85, and de-etiolation det2: Li et al. Plant Cell 9, The morphologic changes are directly related to their deficiency in BR biosynthesis. BRs are also essential in other plants, as demonstrated with studies on a dwarf mutant of Pisum sativum Nomura et al.

In all these mutant plants, use of brassinolide will negate the severe dwarfism. The mechanism by which BR can propagate its effects starts with a cell receptor to interact with a BR. Receptors may be located on the surface of a cell, or within the cell itself Cell-surface receptor kinases activate cellular signal transduction pathways upon perception of extracellular signals, thereby mediating cellular responses to the environment and to other cells.

Some of these RLKs function in growth regulation and plant responses to hormonal and environmental signals. However, the molecular mechanism of RLK signaling to immediate downstream components remains poorly understood, as no RLK substrate that mediates signal transduction has been established in Arabidopsis Johnson et al. Meanwhile, it has been reported that genetic regulation of the brassinosteroid metabolism makes plants highly sensitive to brassinosteroids, and thus an effect of brassinosteroid administration is markedly enhanced Neff et al.

Brassinosteroids bind to the extracellular domain of the receptor kinase BRI1 to activate a signal transduction cascade that regulates nuclear gene expression and plant development. Many components of the brassinosteroid signaling pathway have been identified and studied in detail. However, the substrate of BRI1 kinase that transduces the signal to downstream components remains unknown.


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Brassinosteroids bind the extracellular domain of BRI1 to activate its kinase activity, initiating a signal transduction cascade that regulates nuclear gene expression and a wide range of developmental and physiological processes FIG. The present invention is directed to the discovery of a novel family of kinases that are activated by the binding of brassinosteroid to the brassinosteroid plasma membrane receptor BIN1.

This family of kinases is referred to as BRKs brassinosteroid regulated kinases or BRKs brassinosteroid receptor signaling kinases also referred to as BSK or brassinosteroid signaling kinase. This novel family of kinases interacts with the BIN1 receptors at the plasma membrane and mediates the signaling from the receptor to gene expression. Loss-of-function mutation of BRK3 greatly reduces plant's responsiveness to BRs, and overexpression of the BRKs activates downstream BR responses, including gene expression and cell elongation.

The present invention is based in part on the discovery of a novel family of kinases that are activated by the binding of brassinosteroid with the brassinosteroid plasma membrane receptor BIN1. The present invention provides methods of measuring and determining the signaling generated by brassinosteroids. The present invention also provides methods of modulating the BR signal transduction in a cell. The methods may comprise assaying the kinase activity of a BRK.

The present invention provides nucleic acid molecules encoding the BRK kinase proteins in plants and animals, such as mammals. The present invention provides a novel family of plant kinases involved in the signaling cascades induced by brassinosteroids. The present invention also provides for fusion proteins comprising BRKs. The present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides that encodes a brassinosteroid receptor regulated kinase BRK , wherein the nucleic acid is not derived from Arabidopsis thaliana. The nucleic acid may encode another plant BRK.

The present invention provides polypeptides encoded by the nucleic acids of the present invention. The present invention provides an expression construct comprising a nucleic acid sequence encoding a BRK of the present invention operably linked to a promoter. The present invention also provides a host cell transformed with a recombinant nucleic acid molecule encoding a BRK of the present invention. The host cell may be a plant cell.

The serine at amino acid position three may be phosphorylated. The serine at amino acid position three may be mutated to an alanine, a glutamate or an aspartate. The present invention provides an immunogenic composition, comprising a recombinant nucleic acid molecule encoding a BRK, together with a pharmaceutically acceptable carrier. The present invention provides methods for expressing a BRK of the present invention in a cell. The method may comprise expressing mutant BRK proteins into a cell.

The present invention provides a transgenic plant comprising a nucleic acid encoding a BRK. The present invention provides methods for producing a BRK polypeptide, comprising introducing a nucleic acid encoding a BRK into a host cell and culturing the cell under conditions that allow expression of the BRK polypeptide. The nucleic acid may be operably linked to a promoter. The nucleic acid may be contained in an expression vector. The host cell may be a prokaryotic cell or a eukaryotic cell. The plant cell may be derived from Arabidopsis.

The present invention provides methods for modulating cellular processes of a plant cell comprising introducing a nucleic acid into the plant cell, wherein the nucleic acid encodes a BRK protein. The cellular process may be enhanced as compared to wild-type BRK. The nucleic acid may encode a mutant BRK that cannot function as a kinase. The kinase may be inactivated as a kinase by mutating a phosphorylated serine to an alanine residue. The nucleic acid may encode a constitutively active BRK. The method may further comprise contacting the cell with a brassinosteroid. The present invention provides methods of inhibiting growth of a plant comprising introducing into a cell a ribonucleic acid that hybridizes under stringent conditions to a nucleic acid encoding a BRK polypeptide.

The present invention provides methods for producing a transgenic plant comprising introducing into a plant cell a nucleic acid encoding a BRK. The present invention also provides methods for producing a transgenic plant that has an endogenous BRK knocked-out, or that over-expresses a mutant BRK that cannot function as a kinase.

The plant may exhibit modulated fertility. The plant may be a sterile male.

Plant Germline Development

The plant may exhibit modulated growth. The present invention provides methods for determining the effects of a test substrate on brassinosteroid signaling comprising assaying the kinase activity of BRKs. The present invention also discloses methods for identifying compounds, such as small molecules, that inhibit the kinase activity of BRKs. The present invention also discloses methods for identifying the signaling cascade of BRKs.

These methods may be achieved through use of in vitro and in vivo methods. The methods may be performed using in vitro or in vivo cellular systems. The present invention provides methods for identifying proteins that interact with BRKs. The interaction may be specific to a non-phosphorylated state of a BRK.

The interaction may be specific to a phosphorylated state of a BRK. The interacting protein may be a scaffold protein, a cofactor, or a substrate. The present invention also provides for methods for identifying BRKs in different plant cells. The table summarizes the protein identity, number of unique peptides, and the percentage of protein sequence coverage of mass spectrometry data for the spots numbered in the upper panel. YFP fluorescence images of N.


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Microsomal proteins lanes 1 to 3 were immunprecipitated with anti-GFP antibodies lanes 4 to 6 , and the immunoblot was probed using anti-GFP antibodies or anti-myc antibodies. The right panel shows average hypocotyl length of at least 25 seedlings. Error bars represent standard error. The right panel shows hypocotyl and root lengths average of at least 60 seedlings of wild type Col and brk seedlings grown on various concentrations of BL under continuous light.

Phenotype of light grown three-week-old FIG. Error bars represent standard deviation. Components in the inactive and active states are shown. Components in active states and inactive states are shown. Arrows show positive action, and bar ends show inhibitory action. How the receptor kinases regulate the downstream components has remained an outstanding question. The observed sequence ions are displayed.

Water or ammonium losses are marked with stars. Int: internal ions. Solid underlines show the putative kinase domain, and the dashed lines show the tetratricopeptide repeat TPR domain. The phosphorylated residue S is indicated in the peptide sequence as Sp. Phosphate losses are marked with dots. Protein A bead alone was used as IP control lane 2.

Background

Immunoblots were probed using anti-GFP antibodies. Error bars show standard errors. Stars mark statistically significant differences from Col. The data represent the average from 4 independent biological repeats. Arrows showed spots that show reduced intensity after BL treatment. All three proteins were purified from E. Upper gel image shows autoradiography of 32 P labeling, and the lower image shows total protein stained by Coomassie Brilliant Blue. Figures were generated on line using e-FP browser from at bbc.

Bars in A and C are 5 mm. Seedlings were grown on MS medium in the dark for 4 days. Bar in panel B is 5 mm. Brassinosteroid, or BR, as used herein, refers to a plant growth regulator with a steroid backbone. It is known in the art that brassinosteroids have many functions, such as enhancement of plant growth and plant maturation, and induction of cold and heat resistance. Brassinolide is a type of brassinosteroid. Auxin is a plant growth regulator with an indole backbone that interacts with brassinosteroid signaling.

It is known that some important roles of plant auxins include plant growth and differentiation, formation of flower buds and fruits, and responses to light and gravity. This family of kinases is referred to as BRKs brassinosteroid regulated kinases or BSKs brassinosteroid signaling kinases. This novel family of kinases interacts with the BIN1 receptors at the plasma membrane and mediates the signaling from binding brassinosteroid to gene expression.

Accordingly, as those skilled in the art will appreciate, much of the effects brassinosteroid produce on a plant cell and the overall plant are mediated through this novel kinase family. The effects of brassinosteroids on a plant cell are known in the art. According to the present invention, a BRK may be present or introduced in a plant cell. The plant cell may be derived from any plant. Polypeptides homologous to BRKs may be derived from any eukaryotic cell. While the cDNA were previously reported, expression of a functional protein and the function of the expressed protein was unknown.

A functional equivalent refers to a polypeptide capable of performing the same function, such as the same enzymatic function. For example and not as a limitation, a functional equivalent of a BRK will be able to phosphorylate the same substrate as wild-type BRK. A homolog of a BRK may be from another plant or from an animal. The present invention also discloses identification of a phosphorylation site of the BRK proteins.

The peptide may be obtained by tryptic digestion. In some aspects the present invention provides a BRK containing this peptide in which the serine at the third amino acid of this peptide is phosphorylated by the BRI1 kinase. An antibody refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule having the ability to specifically bind to a particular antigen. Antibodies are well known to those of ordinary skill in the science of immunology. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo.

The subject may be a plant. The subject may be any animal, including a vertebrate. The animal may be a mammal. The subject mammal may be a human, a domestic livestock, a laboratory subject, or a pet animal. For example, an isolated polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell. These steps include the removal of intron sequences by a process called splicing. Examples of conservative substitutions include substitution of one non-polar hydrophobic residue for another e. Examples may include, but are not limited to, standard pharmaceutical carriers such as a phosphate buffered saline PBS solution, water, emulsions, and various types of wetting agents.

A fusion nucleic acid or polypeptide does not necessarily comprise the natural sequence of the nucleic acid or polypeptide in its entirety. Fusion proteins have two or more segments joined together through normal peptide bonds. Fusion nucleic acids have two or more segments joined together through normal phosphodiester bonds. The present invention relates to nucleic acid molecules encoding the BRK kinase proteins.

The present invention also provides nucleic acids encoding BRKs from plants that are not Arabidopsis. BRK proteins and homologs thereof in other plant cells may be identified and utilized. The brassinosteroid receptor has been identified in numerous species of plant. Accordingly, the BRK family of proteins have homologs in other species of plant. By way of example, Tables 1, 2, and 3, illustrate the homology between the coding region of Arabidopsis BRK proteins and the coding region of Vitis vinifera, Glycine max , and Oryza sativa are shown, as well as the kinase domains and the TPR domains.

The present invention provides for nucleic acid sequences that encode a BRK kinase or fragments thereof in other plants. The present invention also provides nucleic acid sequences encoding BRK or fragments thereof from animals, such as mammals. Examples of mammals include, but are not limited to canines, felines, mouse, pig, human, rat, hamster, gerbil, dolphin, whale, seal, cow, sheep, marsupials, and bats. In some instances, the nucleic acids comprise sequences that encode the kinase domain, or mutants thereof. The kinase domain of the BRK may also be mutated.

The present invention provides nucleic acids encoding BRKs or fragments thereof comprising a functionally active kinase domain. A preparation of a polynucleotide encoding a BRK kinase or fragment thereof may be a substantially pure polynucleotide that is free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems. The polynucleotides of the present invention may be in a substantially pure form. By way of example, a nucleotide sequence encoding the kinase domain of a BRK is a subsequence.

The present invention provides nucleic acid construct comprising a nucleic acid molecule encoding a BRK or fragment thereof, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature. The present invention also discloses an expression cassette containing the nucleic acid encoding BRK and control sequences required for expression of BRK.

Control sequences include all components which are necessary or advantageous for the expression of a polynucleotide encoding a BRK protein of fragment thereof of the present invention. Control sequences are operably linked to the nucleic acid encoding BRK. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.

At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide. It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the BRK kinase protein may exist within a population.

Brassinosteroids - Methods and Protocols | Eugenia Russinova | Springer

An allele is one of a group of genes that occur alternatively at a given genetic locus. A gene or a recombinant gene refers to nucleic acid molecules comprising an open reading frame encoding a kinase protein. An allelic variant denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations.

Gene mutations can be silent no change in the encoded polypeptide or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations in a kinase sequence that are the result of natural allelic variation and that do not alter the functional activity of kinase proteins are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding BRK proteins from plant species kinase homologs , which have a nucleotide sequence differing from that of the kinase sequence disclosed herein, are intended to be within the scope of the invention. Also within the scope of the invention are nucleic acid molecules encoding BRK homologs in animals, such as mammals. Nucleic acid molecules corresponding to natural allelic variants and homologs of the BRK cDNA can be isolated based on their identity to the kinase nucleic acid disclosed herein using the cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions as disclosed below.

The present invention also provides for nucleic acids that encode fragments of BRK. The fragments may comprise about 20, 25, 20, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or nucleotides in length. The fragments may encode the kinase domain of a BRK. In addition to naturally-occurring allelic variants of the BRK sequence, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded BRK kinase protein, without altering the biological activity of the BRK protein.

Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention. Alternatively, variant BRK kinase nucleotide sequences can be made by introducing mutations randomly along all or part of the kinase coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for kinase biological activity to identify mutants that retain functional activity, such as kinase activity.

Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques. The present invention includes the polynucleotide sequences encoding BRK from various plant and eukaryotes, such as mammals, as well as fragments and variants thereof. Such probes can be used to detect transcripts or genomic sequences encoding the same or identical proteins. These probes can be used as part of a diagnostic test kit for identifying cells or tissues that misexpress a BRK kinase protein, e.

In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences having substantial identity to the BRK sequences of the present invention. See, e. Sambrook et al. Molecular Cloning: Laboratory Manual 2d ed. Kinase nucleotide sequences isolated based on their sequence identity to the kinase nucleotide sequences set forth herein or to fragments and variants thereof are encompassed by the present invention. In a hybridization method, all or part of a known BRK kinase nucleotide sequence can be used to screen cDNA or genomic libraries. Hybridization may be to a sense strand or to an antisense strand for a nucleic acid encoding a BRK.

An antisense strand is complementary to a strand encoding a BRK or fragment thereof. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor.

Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known kinase nucleotide sequences disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in a known kinase nucleotide sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, , , , , , , , , or consecutive nucleotides of a kinase nucleotide sequence of the invention or a fragment or variant thereof.

Preparation of probes for hybridization is generally known in the art and is disclosed in Sambrook et al. Moreover, stringency conditions and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention are 0. An isolated nucleic acid molecule that hybridizes under stringent conditions to a kinase sequence of the invention may correspond to a naturally-occurring nucleic acid molecule.

The Primary Root of Sorghum bicolor L. Cano-Delgado Prasad, and Tawhidur Rahman. In Stock. A Field Guide to Australian Trees. Medical Herbalism Principles and Practices. Botany for Gardeners. Bonsai With Australian Native Plants. Flora Inside the World of Plants. Item Added: Brassinosteroids. View Wishlist. Our Awards Booktopia's Charities. As the AR can be activated in the absence of hormone, it appears that the conditions may be more restricted than those observed for ER.

Higher doses of ligand actually inhibit proliferation, hi contrast to proliferation, transactivation of AR specific target genes e. Therefore we explored the possibility of cell cycle perturbation by BRs in different cancer cell lines. Flow cytometry was used to evaluate the number of cells in the particular phases of the cell cycle, including SUbG 1 peak detection.

For detection of DNA content analysis, propidium iodide staining was used. Table 4. Histograms of the brassinosteroids treated cells were compared with control untreated cells histograms. Brassinosteroids caused a decrease in percentage of all cell lines in S phase of the cell cycle. DU- cells treated by brassinosteroids 9 or 6 of resulted in decreased level of MDM-2 in all time points Fig. The expression of both phosphorylated and dephosphorylated forms of the retinoblastoma protein Rb decreased after aplication of brassinosteroids 9 or 6 in MCF-7 and MDA-MB cells Fig.

Example 6. Flow-cytometric detection of apoptosis. Several studies have declared that the induction of apoptosis may be cell cycle-dependent. Therefore, in our next series of experiments, we tested if brassinosteroids 9 or 6 cause apoptosis of the breast and prostate cancer cells via cell cycle blockage. From this reason we carried out DNA cell cycle analysis by flow cytometry based on detection of endonucleolytic DNA degradation that results in extraction of low molecular weight DNA from the cells.

Flow cytometry, used as a conventional technique for detection of a particular form of cell death, showed an appearance of a SUbG 1 peak in the hypodiploid region of the cell cycle-related DNA histograms. The observation of apoptosis after brassinosteroid treatement is important because the molecular analyses of human cancers have revealed that cell cycle regulators are frequently mutated in most common malignancies.

Cells were visualized using fluorescence microscopy and compared with control cells. TUNEL staining achieved to confirm apoptosis in all cell lines. Ctrl 6 h , Ctrl 12 h , Ctrl 24 h -untreated controls; 9 6 h , 9 12 h , 9 24 h - cells trested with compound 9; 6 6 h , 6 12 h , 6 24 h - cells trested with compound 6. DMSO was used as a vehicle for controls. The lysates were collected into microfuge tube and incubated on ice for 1 h. Cells were incubated 60 min at 4 0 C under time by time shaking in protein exctaction buffer.

The blot was incubated overnight in 4 0 C with appropriate primary antibody that detects the protein epitope of interest and finally washed in PBS with 0. The membrane was washed in PBS with 0. The proteins were detected using a chemiluminescence detection system Amersham Biosciences, Vienna, Austria according to the protocol provided by the manufacturer. Equality of loaded proteins was confirmed by Ponceau S membrane staining Sigma, St. The experiments were repeated three times. The protein expressions in treated cells were compared to untreated controls. Western blot analysis was used to detect changes in apoptosis related protein expression in breast and prostate cancer cell lines.

To monitor changes over the 24 h treatment, we collected the cells after 6, 12 and 24 h treatment with BRs in concentration IC 5O evaluated by MTT assay. Changes in apoptosis related protein expression after treatment with BRs are shown in Fig. LNCaP cells showed an increased level of pro-apoptotic Bax after the 9 treatment after 6 and 12 h and decreased expression of pro-apoptotic uncleaved protein Bid after 6 and 12 h treatment with both BRs. This decreased expression of Bid can indicate a cleavage of the protein after treatment by BRs.

Brassinosteroid 9 treatment of LNCaP cells induced degradation of caspase-3 into its cleaved fragments after all time points 6, 12 and 24 h. Brassinosteroid 6 resulted in an activation of caspase-3 after 24 h treatment of LNCaP cells. On the other hand, caspase cleavage was not found in DU- cells. In this study we have shown that compound 9 treatment to LNCaP resulted in significant decrease in the levels of anti-apoptotic Bcl-2 protein and increase in the pro-apoptotic Bax protein.

Brassinosteroid Biochemistry

Moreover, a degradation of procaspase-3 into cleaved fragments was detected after all time points of treatment with 9 and after 24 h treatment with 6 in LNCaP cells. This findings whose to iniciation apoptosis changes in LNCaP cells. It has been known that the execution mechanism of apoptosis is mediated by caspase cascade activation Budihardjo et al.

Cell Dev. Life Sci. These results confirm that brassinosteroids 9 and 6 can support apoptosis with caspase-3 activation and modulations in BcI -2 family proteins in cell lines derivated from prostate carcinoma. In breast cancer cell lines, Western blot analysis showed no significant changes in expression of Bcl-2 family protein nor caspase-3 activation after treatment with brassinosteroid 9 and 6. Preparation process: The powdered substances mentioned are pressed through a sieve of mesh width 0. Portions of 0. Example 9 Soft Capsules. Portions of in each case 0. Ref country code : DE. Ref document number : Country of ref document : IL.

Country of ref document : EP. Country of ref document : US. The present invention relates to natural brassinosteroids of general formula I , wherein R is CH2 or O-CH2 group, R2 is hydrogen or hydroxyl, R3 is hydroxyl, R24 is alkyl or alkenyl, which are selected from the group consisting of methyl, ethyl, propyl, isopropyl, methylen, ethylen and propylen, and R25 is alkyl selected from the group consisting of methyl and ethyl, and a pharmaceutically acceptable salt thereof, for use for treating hyperproliferation, treating proliferative diseases and reducing adverse effects of steroid dysfunction in mammals.

The present invention also provides methods capable to arrest of the cell cycle by natural brassinosteroids resulting in apoptotic changes in cancer cells. More specifically, the present invention relates to use for treatment of the adverse effects of hyperproliferation on mammalian cells in vitro and in vivo, especially treatment of hyperproliferative diseases in mammals by administering compositions containing natural brassinosteroids. This invention also describes new use for treating consisting in a new therapeutic way for modifying cell viability of human breast and prostate cancer cells.

Natural brassinosteroids for use for treating hyperproliferation, treating proliferative diseases and reducing adverse effects of steroid dysfunction in mammals, pharmaceutical composition and its use Field of invention This invention relates to natural brassinosteroids and their derivatives for use for the inhibition of hyperproliferation in mammalian cells, for treating proliferative diseases in mammals, and for regulation of the adverse effects of steroid disfunctions in mammalian cells and mammals. Background of the invention Brassinosteroids are steroid plant hormones with important regulatory roles in various physiological processes, including growth, differentiation, root and stem elongation, disease resistance, stress tolerance and senescence Bajguz et al.

Summary of the Invention The object of this invention are natural brassinosteroids of general formula I. Natural brassinosteroid of the general formula I. R 25 is alkyl selected from the group consisting of methyl and ethyl, and a pharmaceutically acceptable salt thereof, for use for treating hyperproliferation, treating proliferative diseases and reducing adverse effects of steroid dysfunction in mammals.

Natural brassinosteroids of the general formula I according to claim 1 for use for treating of proliferative diseases of mammals characterised by administration of an therapeutically effective amount of natural brassinosteroids to the mammal's hyperproliferating cells in need of such treatment, wherein the natural brassinosteroid derivative is selected from the group consisting of the following compounds: castasterone, homocastasterone, epibrassinolide, dolichosterone, 2-deoxycastasterone, typhasterol, teasterone, 3-oxoteasterone, cathasterone, 6-deoxotyphasterol, 3- dehydrodeoxoteasterone, homotyphasterol, homoteasterone, homodolichosterone, methylcastasterone, methyldolichosterone, 2- deoxymethyldolichosterone, and 3-epideoxy methyldolichosterone.

Natural brassinosteroids according to any of the preceding claims, wherein R 2 , R 3 , R 24 , and R 25 have independently at each occurrence R or S configuration. Natural brassinosteroids for use for inhibiting cell proliferation in mammals comprising administration of a therapeutically effective amount of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof together with a pharmaceutical carrier.

Natural brassinosteroids of the general formula I according to any one of claim 1 to 3 for use according to any of the preceding claims as growth regulators in animal and human tissue cultures for regulation of proliferation and morphogenesis. Natural brassinosteroids of the general formula I according to any one of claims 1 to 3 for use for inhibiting cell proliferation in mammals comprising administration of a therapeutically effective amount of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof together in combination with usually used cytostatics, such as mitoxantrone, cis-platinum, methotrexate, taxol, or doxorubicin.

Natural brassinosteroids of the general formula I according to any one of claims 1 to 3 for use for inducing apoptosis in mammalian cells comprising administering to a subject a therapeutically effective amount of natural brassinosteroid of the general formula I, according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof together with a pharmaceutical acceptable carrier. Natural brassinosteroids of the general formula I according to any one of claims 1 to 3 for use for treating cancer in mammals comprising administration of a therapeutically effective amount of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof to a subject together with a pharmaceutical carrier.

Natural brassinosteroids of the general formula I according to any one of claims 1 to 3 for use for inhibiting of cancer according to claim 8, wherein cancer is of prostate, endometrium or breast origin. Natural brassinosteroids of the general formula I according to any one of claims 1 to 3 for use for regulation od adverse effects of steroid disfunctions in mammals cells comprising administration of a therapeutically effective amount of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof together with a pharmaceutical carrier, wherein the steroid disfunction is osteoporesis, cholesterol metabilism defects, Alzheimer disease, Huntington disease, steroid-induced cataracta, deficiency of P oxidoreductase, men infertility amd disorder of sexual behaviour and differentiation.

Pharmaceutical composition comprising an effective amount of natural brassinosteroids of the general formula I according to claim Pharmaceutical composition according to the claim 14 for use for treatment of mammalian cells and human beings. Natural brassinosteroids intended for use when treating hyperproliferation, further for treating proliferative diseases and reduction of unfavorable effects of steroid dysfunctions in mammals, pharmaceutical compositions containing thereof and their. USA1 en. EPA2 en. CZB6 en. ILD0 en. WOA2 en. USB2 en. WOA3 en. CZA3 en. KRB1 en. Mukherjee et al.

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Brassinosteroids: Methods and Protocols Brassinosteroids: Methods and Protocols
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