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WO2002031103A1 - Dispositif et procede de regulation de l'expression genique a champ electromagnetique cyclique - Google Patents

Dispositif et procede de regulation de l'expression genique a champ electromagnetique cyclique Download PDF

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Publication number
WO2002031103A1
WO2002031103A1 PCT/KR2001/000896 KR0100896W WO0231103A1 WO 2002031103 A1 WO2002031103 A1 WO 2002031103A1 KR 0100896 W KR0100896 W KR 0100896W WO 0231103 A1 WO0231103 A1 WO 0231103A1
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WO
WIPO (PCT)
Prior art keywords
cyclic
gene expression
sequence
dna
magnetic field
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PCT/KR2001/000896
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English (en)
Inventor
Seok-Kun Lee
Yeon-Sook Kim
Yun-Woo Lee
Je-Geun Chi
Original Assignee
Lee Seok Kun
Kim Yeon Sook
Lee Yun Woo
Chi Je Geun
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Filing date
Publication date
Application filed by Lee Seok Kun, Kim Yeon Sook, Lee Yun Woo, Chi Je Geun filed Critical Lee Seok Kun
Priority to AU2001262764A priority Critical patent/AU2001262764A1/en
Publication of WO2002031103A1 publication Critical patent/WO2002031103A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J19/087—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J2219/0803—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy
    • B01J2219/085—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy creating magnetic fields
    • B01J2219/0854—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy creating magnetic fields employing electromagnets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J2219/0803—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy
    • B01J2219/085—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy creating magnetic fields
    • B01J2219/0858—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy creating magnetic fields employing moving elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J2219/0803—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy
    • B01J2219/085—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy creating magnetic fields
    • B01J2219/0862—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electric or magnetic energy creating magnetic fields employing multiple (electro)magnets
    • B01J2219/0867—Six or more (electro)magnets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J2219/0873—Materials to be treated
    • B01J2219/0877—Liquid

Definitions

  • the present invention relates to an apparatus for regulating gene expression by using cyclic magnetic field and the method thereof, or more particularly, to an apparatus for regulating gene expression by using cyclic magnetic field and the method thereof, which comprises the steps of determining the sequence of a DNA or RNA base sequence considered to have biochemical activity obtained through analysis which predicts activation and the path of movement of free electrons induced between the base pairs in hydrogen bond of a DNA or RNA double helix in natural condition or in the presence of cyclic magnetic field, or tlirough DNA or RNA database search; and then irradiating a cyclic magnetic field specific to said sequence.
  • the genetic information carried by DNA of an organism consists of such bases as A, C, G, and T, and the respective sequences of these bases in their arrangement are known to send off specific signals in order to react with various substances in the relevant protein or substrate.
  • the genomic DNA the very cores of cells, is largely classified into intron DNA and exon DNA. It is known that the base sequence of an intron DNA elongates and becomes the more complicated, as evolution progresses, and it is presumed that an intron DNA, having a promoter region and other regions for an enhancer and a silencer, issues its DNA signals, which keep under control the time for the relevant gene's expression, its rate, amount, etc.
  • the range of the frequency can vary from specific case to case, and it may be raised to 10 14 Hz or more.
  • the intensity and the frequency of an electromagnetic field were calculated as functions of the quantum genetic target and the mass of the structure bonded thereto. For instance, homeobox, RNA, proteins, hormone, neurotransmitter, water molecules, calcium and trace elements such as genes (specific DNA fragments) as well as protons and electrons are included.
  • USP 5,919,679 describes a method and a device to change the ionic bond by the use of magnetic fields, h other words, this patent provides a method and means of changing or otherwise influencing the ionic bonds in such systems as contains unhydrated ions, including cells and other biological systems, by the use of magnetic fields.
  • the method is one which is to induce a particular magnetic reaction between ions and the molecules bonded therein, and is related to one which is to change the intensity and cycle of change for the static and sinusoidally varying magnetic fields, which makes a pair with its directionality.
  • IPR ion parametric resonance
  • the present invention provides a method and device to generate cyclic magnetic fields with the use of a plurality of sources, and thereby to regulate the expression of genes peculiar to the base sequences of specific DNA or RNA.
  • Fig. 1 illustrates the polarity, anticipated between a DNA base pair in natural conditions or in case of applying electromagnetic fields in the perpendicular direction to the hydrogen bond between the DNA base pair.
  • Fig. 2 is a diagram for the provisional signals based on the path of movement of electrons in a DNA helix structure.
  • Fig. 3 shows the classified forms of DNA fragments according to the path of movement of free electrons, predicted according to the method for DNA analysis based on the polarity between a DNA base pair.
  • Fig. 4 shows the path of movement of electrons as predicted in the region of a promoter of a human p53 gene (Genbank Accession No. AF082338) based on the concept of polarity between a DNA base pair.
  • Fig. 5 is a bloc diagram of the structure of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention.
  • Fig. 6 A is a diagram illustrating an example of the oscillator circuit of the driver of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention.
  • Fig. 6B is a diagram illustrating an example of the oscillator circuit of the driver of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention, which shows a relay reinforcement circuit necessary to maintain the nodes of Cl and of Al in CR1 of Fig. 6 A.
  • Fig. 7 is a diagram illustrating an example of the control circuit of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention, which is a switching circuit for a continuous run until TRn tlirough sequential switching operation.
  • Fig. 8A is a plain view of the magnetic-field reactors of a horizontal type, which illustrates the basic structure thereof in the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention.
  • Fig. 8B is a plain view of the magnetic-field reactors of a vertical type, which illustrates the basic structure thereof in the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention.
  • Fig. 8C is a partial side view showing one of the electromagnets positioned for multi-polarity in the magnetic-field reactors of a vertical type as illustrated in Fig. 8B, and the outer frame to which said electromagnet is connected.
  • Fig. 9A illustrates an example of sequence signals applied from the microprocessor of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention, which shows the sequence signals, reversed for distinguishing T and C from A and G, respectively, while changing the lengths of time with the same voltage while dividing the cases of A or T from those of G or C.
  • Fig. 9B illustrates an example of sequence signals applied from the microprocessor of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention, which shows the sequence signals, reversed for distinguishing T and C from A and G, respectively, while changing the voltage with the same length of time while dividing the cases of A or T from those of G or C.
  • Fig. 10A is a plain view of a rectangular shielding plate against cyclic magnetic fields of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention.
  • Fig. 1 OB is a plain view of a snail-shape shielding plate against cyclic magnetic fields of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention.
  • Fig. 11 A illustrates an embodiment of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention, which shows the sequence of a T3 promoter and the signal according to the path of movement of free electrons in said sequence.
  • Fig. 11B illustrates an embodiment of the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention, which shows the results of electrophoresis showing the effects to transcription by T3 RNA polymerase on account of irradiation of a specific cyclic magnetic field to the T3 promoter by using the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention.
  • Fig. 11C is a graph showing the results of Fig. 11B, measured by a densitometer.
  • Fig. 12 is a graph, which shows the effects to the expression of mouse alkaline phosphatase on account of irradiation of a specific cyclic magnetic field to the alkaline phosphatase promoter by using the apparatus for regulating expression of genes using cyclic magnetic fields according to the present invention.
  • the numbers are used to represent zones, units, circuits, or parts of the present invention as follows: 100 for negative zone; 102 for positive zone; 104 for (Py-Pu)n rich zone; 106 for well polarized zone; 20 for a driver; 21 for a power circuit; 22 for a wave generator circuit; 23 for a control circuit; 30 for a magnetic-field reactors; 40 for a microprocessor; 70 for an electromagnet; 71 for an outer frame; 72 for a magnetic-field shielding device; 73 for a platform; 90 for a rectangular magnetic- field shielding device; and 91 for a snail-shape magnetic-field shielding device.
  • the present invention consists of the components to be described below, but, prior to entrance into the detailed description of the invention, its theoretical foundation is to be given first.
  • a DNA chain is in a firm construction, in a single chain, by means of such a covalent bonding as the phosphate diester bonding between ribose and phosphate, or the glycosidic bonding between ribose and base, but it forms base pairs together with the opposite DNA chain by virtue of a hydrogen bonding.
  • a hydrogen bonding it can be expressed, is not in fact in a state of sharing electrons but in a state, in which the molecules are in a strong activation by the phenomenon of resonance close between themselves, hi such a circumstance, it is well known, the protons in the atoms of hydrogen particularly come under the phenomenon of a mighty resonance with magnetic fields.
  • the present inventors surmised that, if a base pair was irradiated with electromagnetic fields perpendicularly to the direction of its hydrogen bonding, the protons' resonance would increase and that, at that time, according to Fleming's left- hand rule, the electrons' movement would begin in the direction opposite to the that in which the protons moved, that is, opposite to the direction of electric current movement.
  • a and T will make a pair and G and C another, of the four bases in a DNA, and because T and C among the base pairs of purine (A or B) and pyrimidine (T or C) of the four are with relatively much negative charge, A and G will be of positive charge, as is seen in Fig.
  • a pyrimidine-purine repeats shows the pyrimidine-purine repeats, and in case a pyrimidine occupies 5' of a pyrimidine-purine repeat and a purine 3', a chi box is formed, which is shown in boxes of a thick line.
  • a phenomenon of electrons' cyclic movement like this occurs when a base sequence of pyrimidine and purine continues, and as such a circulatory movement of electrons between DNA chains is anticipated in the case of a base sequence of TA, CA, TG, CG, etc., such symbols as are given in Fig. 2 have been devised to indicate the polarity of the structure of a DNA base pair sequence on the basis of the state of hydrogen bonding between the electrons and the direction of their movement.
  • the pyrimidine-purine repeats viz. TA, TG, CA, CG, etc. are shown as D
  • a simple DNA segment consists only of a Chi-box; while a negative DNA segment as a whole consists chiefly of negative charges; a positive DNA segment, as a whole, chiefly of positive charges; a composite DNA segment, having a number of pyrimidine-purine repeats in the same segment and is distributed with negative and positive bases in turn; and a composite DNA segment, having a number of pyrimidine-purine repeats in the same segment, does contain a reverse Chi-box.
  • the DNA sequence in the promoter region of a human (p53) gene is schematized, which can be divided in the positive zone 100, the negative zone 102, the well polarized zone 106, and the purine-pyrimidine rich zone 104, and since, when the base sequence in the zone rich with purine-pyrimidine repeats is activated the expression of the relevant hereditary material is tended to increase, the zone is focused as an area for intensive research of the region.
  • the present inventers have inquired into the DNA bonding areas of the transcription factors of a variety by such methods as given above for analysis of DNA, and as a result they have found that such areas are mostly of meaningful structures, and, in especial, analyses relative to the base sequence in the target DNA binding of the restriction enzymes have been considered very meaningful. Furthermore, the present inventors have also performed researches in some simple in vitro transcription without any double structure or complex elements in a cell, in which DNA produced by the use of PCR was used to avoid the multiplicity of a 3-D structure of DNA. The details are given in the embodiment examples below.
  • the methods for determining the sequence which seems active toward the movement paths of free electrons in a DNA can be applied as it is to the RNA which consists of a single strand, too, because it is in any case a phenomenon that occurs in the DNA of a single axis, h other words, although in RNA the T (thymine) of DNA is substituted by U (uridine) in its bases, both do the hydrogen-binding with A (adenine), and U is different from T only by its lack of one methyl group, and so U also has more negative charges than A has, wherefore, it is easily supposed that movement of electrons can take place in the same RNA axis. Accordingly, the above-given methods for analysis of DNA base sequence can similarly be applied to that of RNA also.
  • the method of the present invention for regulation of the expression of genes by irradiation with cyclic electromagnetic fields is characterized in that it comprises a step of determining a number of DNA base sequences regarded as having biochemical activity either by an analysis predicting the movement paths of free electrons induced between the base pairs hydrogen-bonded on the double helix of DNA or RNA naturally or in the presence of cyclic electromagnetic fields, or by an inspection of DNA or RNA databases; and a step of irradiating a biological system with a cyclic electromagnetic field peculiar to the above sequence groups, cyclically at a certain interval of time.
  • FIG. 5 An example of the apparatus for regulation of the expression of genes by the use of cyclic electromagnetic fields, as is seen in Fig. 5, is characterized in that it consists of a microprocessor 40, which receives the input of the sequence which is regarded as having biochemical activity toward the gene which is intended to express and outputs signals of such sequences; a driver 20, which receives such signals from said microprocessor and accordingly outputs pulsating driving signals; and a reactor 30 which, equipped with electromagnets, generates cyclic electromagnetic fields by virtue of said magnets in response to the driving signals received from said driver.
  • Fig. 4 is a rough block diagram to show an example of such construction of the apparatus of the present invention.
  • the microprocessor 40 as is shown in Fig.
  • the driver 20, as is seen in Fig. 5, has got such a characteristic that it consists, for instance, of a power circuit 21 which receives input of power from an outer source, rectifies it and outputs a direct current; a oscillator 22 generates the sine wave of a certain frequency; and a control circuit 23 which receives supply of power from said power circuit 21, receives said sine wave signal from said oscillator 22, forms pulsating driving signals in response to the sequence signals input from said microprocessor 40, and outputs the thus generated pulsating signals.
  • a power circuit 21 which receives input of power from an outer source, rectifies it and outputs a direct current
  • a oscillator 22 generates the sine wave of a certain frequency
  • a control circuit 23 which receives supply of power from said power circuit 21, receives said sine wave signal from said oscillator 22, forms pulsating driving signals in response to the sequence signals input from said microprocessor 40, and outputs the thus generated pulsating signals.
  • said power circuit 21 being a supplier of the power required by the magnetic field driver, is a apparatus to lower the A. C. power of 110 ⁇ 220v, 60 Hz, to some appropriate voltage, and converts it into D. C. if necessary.
  • a circuit designed for adjusting the voltage and quantity of power in accordance with the capacity of the magnetic field reactor and such a rectification circuit as a bridge can be used.
  • an overloading is apt to take place on said magnetic field reactor, it may be the case that an adequate safety resistance is necessary and a cooling device is also required to prevent overheating.
  • said oscillator 22 is a device to generate the sine wave of a certain frequency.
  • said oscillator 22 can be made either into a oscillator which uses a simple relay and a condenser, or into one having, in extra, a relay- reinforcement circuit for maintenance of a node between Cl and Al of CR1 of 6 A shown in 6B.
  • said control circuit 23 is a device which receives power from said power circuit 21, sine wave signals from said oscillator 22, and generates pulsating driving power signals in accordance with the sequence signals received from said microprocessor 40 to output the thus generated signals.
  • Fig. 6 showing an example of the circuit which can be used for a control circuit 23, is a diagram, in which the switching circuit can work up to TRn in succession by the successive operations of the individual switches. According to this circuit, when a generation is achieved of full frequency, TR1 is worked to fill Cl with power and work TR2 with the mechanism of Cl, and it is possible this way to work up to TRn in succession.
  • the driver 20 can be used in two or more in number to meet the number of active sites of the subject gene.
  • the reactor can at this time be worked in succession or simultaneously.
  • the control circuit 23 can have a safety circuit attached to it.
  • the safety circuit can contain a fuse system for both inner and outer power sources, a fuse box (safety cut-off) for prevention of overheating from leaks of said reactor 30, an automatic breaker to avoid leaks due possibly to the overloading of all the electronic equipment and devices, etc.
  • To said control circuit 23 can be attached an adjustment device, in addition, for control of an electrical motor in case one with a decelerator has to be used as said driver 20 necessarily grows very large, h case said driver 20 gets to be more than one in number, say, as many as about 5—10, extra control circuits may also be required to work them in succession.
  • the reactor 30 is characterized in that it consists, as shown in Figs. 8A, 8B, and 8C, for instance, of a platform 73, on which the biological system for expression is placed; an outer frame 71, which contains said platform, is at least twice as thick as said electromagnets, and is made mainly of non-carbon steel; electromagnets with at least two or more poles, placed inside said outer frame 71 with said platform at the center; magnetic-field shielding plates 72 placed between the poles of said electromagnets; and a position-adjusting device (not shown in the drawings) to see to it that said biological system is placed precisely at the desired position by moving said platform 73 or said outer frame 71.
  • said magnetic field reactor 30, said platform 73 being a stand or plate on which to put said biological system for irradiation with electromagnetic fields, can be of any shape, made of any material stuff, if only it does not interfere with the irradiation with magnetic fields.
  • the platform 73 connected with the following adjustment device, can be made to move up and down and to left and right. Further, it can turn round at level or cubically.
  • said magnetic field reactor 30, said outer frame 71 has thickness just enough to concentrate the magnetic field at the center, and is connected with the surrounding magnets. The outer frame 71 must be far thicker than the cores of the magnets.
  • said outer frame 71 serves as a mediator to assure of generation of a magnetic field at the U-ends as in the case of a horse-shoe magnet, and if its thickness is made too thin it will fail to generate a field of a sufficient magnetic field.
  • the size of said outer frame 71 can vary. For instance, a reactor with an outer frame of 20cm ⁇ 3m in diameter can be manufactured.
  • said electromagnets 70 are of at least two or more poles and positioned inside said outer frame 71 with said platform at center, and they receive driving signals from said control circuit 23 of said reactor 30 alternately to generate cyclic magnetic fields with a multiple polarity, say, of 10 poles.
  • said magnets must be of two or more poles is that they cannot be otherwise if they are to shoot cyclic electromagnetic fields perpendicularly to the hydrogen bonds between the base pairs on a DNA or RNA double helix.
  • they are with about 10 poles, the number corresponding with that of the bases that take part in a complete turn round of a DNA or RNA double helix, h other words, they had better be about 6-14, or more preferably 8 ⁇ 12 poles, the number equal to that of the bases required for a complete turn round of a double helix in ordinary DNA structure.
  • the electromagnets 70 include ones of non-carbon steel, with enamel coil wound on.
  • a number of such magnets 70 preferably about 10, are set up in attachment to said outer frame 71, and they can be arranged radially with said platform 73 at the center, either a plane or a flat type, so that their axes may be kept at level (Fig. 8A) with or perpendicularly (8B, 8C) to the ground; or else, arranged spherically radially in a 3-di-mensional type with said platform 73 at the center so that the magnets' axes may face the spherical surface of said outer frame in a normal line.
  • Fig. 8 shows an example of a horizontal type of a magnetic field reactor, in which said magnets are arranged levelly, with 10 magnets 70 in a horizontal and radial arrangement.
  • Fig. 8B is an example of a vertical type of a magnetic field reactor, in which a number of magnets are vertically arranged, showing 10 of them 10 in a vertical and radial arrangement.
  • Fig. 8C is a partial view of a vertical section to show how one 70 of the vertically arranged magnets in Fig. 8B is attached to the lower end of said outer frame 71.
  • the metal used in generating the electromagnetic field must need to be non-carbonic steel.
  • magnetic-field shielding devices 72 mean such separators as shown in Figs.
  • a cylindrically modified magnetic field shielding devices 72 can also be used.
  • said adjustment device (not shown in the drawings) is connected with said platform 73 and said outer frame 71, and, set up in the lower end of said reactor, moves the whole of said outer frame 71 or only said platform 73 to left and right or upward and downward to see that said biological system is placed exactly at a particular place of said reactor.
  • said adjustment device turns the biological system for irradiation little by little in the clockwise or anticlockwise direction at each cycle of irradiation, or turns the whole of said outer frame 71 in relation to the biological system under irradiation.
  • the adjustment device is one that can move the object of irradiation not merely horizontally but in 3-D directions also so that the biological system under irradiation can get exposed to magnetic fields on its total 3-D surfaces.
  • adjustment device can be used all kinds of motors, cylinders, racks-and- pinions, belts, etc., in use in the field of the industry.
  • another such means of 3-dimensionally irradiating biological systems with cyclic magnetic fields is possibly achieved by using any device, in which a number of magnetic-field generators with magnetic field reactors arranged 3- dimensinally are so arranged as to irradiate the objects with cyclic electromagnetic fields at the command of a computer.
  • an adjustment device can be resorted to, which can grasp the polarities of appropriate DNA base sequences from several loci and irradiate these loci cyclically with cyclic magnetic fields of 5 ⁇ 20 kinds at certain intervals of time.
  • the reactor 30 can get extremely heavy as its size grows, and by this reason it may become necessary to provide an auxiliary support frame to hold it, while such an auxiliary support frame can be operated by an electrical deceleration motor, and for this operation an adjustment device under the control of a personal computer software can be added, too.
  • the magnetic field operator consisting of 10 poles is only given as an example to describe the present invention, and naturally the present invention shall not be limited or confined to such by any means, for according to the desired use and to the degree of detailed control of the produced magnetic fields to be generated, one of fewer poles than 10 or of more than that can be put to use.
  • magnetic-field shields which can include inner shields to minimize the mutual interferences of the many magnets with one another. Description of such a magnetic-field shielding device 72 may be found elsewhere above. Ordinarily, for such shields to separate magnetic objects iron nets are in use, but because such magnetic substance as iron nets can possibly cause decrease of the resonance produced by cyclic magnetic fields with its target if such are used, it may prove profitable for an inner shield to use non-magnetic substance to stop irradiation outward from the magnetic field reactor.
  • the magnetic field shields can include outer shields 90, 91 in addition to prevent such relatively strong alternating electromagnetic fields from emission out of the area of said magnetic field reactor to harm the operator or other unintended biological systems.
  • outer shields such magnetic metals as iron nets, for use in ordinary shields, can also be made use of.
  • shields can also serve to decrease noise from outside from indeterminate electromagnetic field sources which may interfere with the particular cyclic signals in the present invention.
  • Fig. 10 illustrates an example of outer magnetic field shields in the present invention.
  • Fig. 10A shows an example of a rectangular shield 90 and Fig. 10B, of one 91 in the shape of a snail. According to Fig. 9, both are made in a manner so as to cover up the entire magnetic field reactor, the doors are prepared in a round-about way to block off leak of magnetic fields direct from the shield to outside.
  • the material used here is magnetic metal like an iron plate. No door is shown in the above drawing, but it is quite natural that the plate can be equipped with a door.
  • Fig. 9 A shows that the sequence is a signal, where the cases in which said sequence is A or T are distinguished from where it is G or C, rendered at the same voltage but in different lengths of time, and reversed so that T and C may be distinct from A and G, respectively.
  • Fig. 9B shows, on the other hand, that the sequence is a sequence signal, where the cases in which said sequence is A or T are distinguished from where it is G or C, rendered at different voltages but in the same length of time, and reversed so that T and C may be distinguished from A and G, respectively, hi other words, Fig. 9A shows the way to distinguish A, T, G and C from each other by the lengths of time and reversion of the magnetic field, while Fig. 9B the way to distinguish them each by the voltage and reversion of the magnetic field.
  • the control circuit 23, which receives said sequence signals from said microprocessor 40, as described above, now receives power from said power circuit 21 and sine wave from said oscillator 22, and then forms pulsating driving power signals from said sequence signals to supply them on each magnet 70 of said reactor.
  • said driving power signals specific to the DNA or RNA base sequence regarded as seemingly having biochemical activity are supplied to each magnet 70 of said reactor.
  • said power circuit 21 receives power from an outer source and converts it to a direct current. For instance, it performs a function of receiving input of an alternating current of 110-220V, 60Hz from an outer source and converting it to a direct current.
  • the power circuit 21 adjusts the voltage and amount of power according to the requirement by said reactor 30. Generally, said voltage and amount of power are so adjusted that the strength of the magnetic field at the central area of said reactor 30 will be kept at 5-100 gausses, but not necessarily restricted to this range.
  • said oscillator 22 does the function of generating sine wave signals of a certain frequency.
  • the driving power signals which said control circuit 23 generates are electrical signals of certain frequencies, which cause said magnets 70 arranged with a multiplicity of poles, say, 10 poles, in said reactor 30 to generate cyclic and alternating magnetic fields. They can be a sort of a pulsating direct current, combined of said sine wave signals supplied by said oscillator 22 and the direct current supplied by said power circuit 21 in accordance with said DNA or RNA sequence signals considered biochemically active. As shown in Fig.
  • the driving power signals output to said magnetic field reactor are generally preferred to change their cycle about 20 to 24 times in 5-7 seconds, but, as circumstances require, the cycle can be increased to as many as said magnets, or, for instance, the reluctance of the enamel coils, does permit.
  • said driving power signals generated by said control circuit 23 and specific to a DNA or RNA sequence, are supplied to said magnets 70 arranged with many poles, say, 10 poles, at said reactor 30, said magnets 70 generate magnetic fields, specific to said DNA or RNA sequence according to said driving power signals.
  • the use of the apparatus of the present invention to regulate the expression of genes comprises a step of inputting to a microprocessor of the device such sequences determined to be biochemically active through an analysis for prediction of the paths of the movement of free electrons induced between the base pairs which are hydrogen- bonded on a DNA or RNA double helix, naturally or in the presence of cyclic magnetic fields, or through an inspection of DNA or RNA databases; a step of so moving the outer frame or platform of the device by operation of the adjusting device that the biological system for irradiation with cyclic magnetic fields may be placed precisely at the desired position; and a step of applying the reactor with such driving power signals specific to the sequence from the control circuit so that irradiation with cyclic magnetic fields can be performed for a certain length of time.
  • the step of determining a sequence considered having biochemical activity through an analysis for prediction of the paths of the movement of free electrons induced between the base pairs which are hydrogen-bonded on a DNA or RNA double helix, naturally or in the presence of cyclic magnetic fields, or through an inspection of DNA or RNA databases does mean a step of determining the sequence considered to be the protein binding site, on the basis of a selection of regions where cyclic electron movement is concentrated, by the method for analysis described in detail in [the theoretical basis relative to the present invention] above, and also on the basis of such relevant regions which, in especial, have left and right symmetry and the like. Examples of such DNA or RNA analyses are given in Figs. 1, 2, 3, and 4.
  • Fig. 1 illustrates a prediction of the polarity between the base pairs on a DNA double helix, consisting of a GTCGACA forward sequence.
  • Fig. 2 is a drawing showing, schematically, the movement paths of free electrons, while Fig. 3 shows the structure of a DNA fragment, schematized by polarizing a base sequence.
  • Fig. 4 an example of schematization of DNA information, shows the base sequence of human p53 gene's promoter region (GenBankTM Accession No. AF082338), distinguishing the region rich in pyrimidine-purine repeats.
  • the step of determining a sequence considered having biochemical activity includes, the step of selecting DNA or RNA sequences already known to have biochemical activity by searching DNA or RNA data base, for example, GenBankTM.
  • the step of irradiation means a step of irradiation of the subject biological system with cyclic magnetic fields specific thereto by the use of a device for generating cyclic magnetic fields.
  • the device for generating cyclic magnetic fields can include the device of the present invention for regulation of the expression of genes, which, as has been described all above, consists of said microprocessor which receives input of the sequence considered having biochemical activity in relation to the subject biological system for expression, and outputs sequence signals in accordance with said sequence; a driver which outputs pulsating driving power signals according to the received signals; and said reactor which generates cyclic magnetic fields by electromagnets in accordance with the driving power signals input from said driver.
  • a cyclic magnetic field specific to said sequence is meant one, for instance, which causes the control circuit to generate the driving power signals, distinguishing A, T, G and C, and outputs them to said electromagnets arranged in said reactor with a multiple polarity in succession, thus itself being cyclic and yet capable of distinguishing A, T, G and C.
  • the method for distinguishing A, T, G and C is, as shown above, first by the length of time and reversion of the driving power signals; that is, as each driving power signal contains 1-2 pulses, power is input with 1-2 pulses a time in the case of A or T, and 2-4 a time in the case of G or C (See Fig. 9).
  • A, T, G and C are to be distinguished by the voltage of power and reversion of the driving power signals, higher voltage of power is input in the cases of G or C than in the cases of A or T (See Fig. 9B). Accordingly, the magnetic fields thus generated by the driving power signals are also the cyclic magnetic fields specific to the sequences which distinguish A, T, G and C.
  • the subject biological systems include all such systems as have a DNA double helix.
  • they include viruses, bacteria, yeast, fungi, plants, and all kinds of animals including humans.
  • If one is a biological system which has a DNA double helix it is all right regardless of whether the double helix is of a single or a double strand.
  • the biological systems which have a DNA double helix but all sorts of biological systems can be included in the range, if they only contain a molecule, in which there is a hydrogen bond and movement of free electrons can take place cyclically.
  • biological systems which have a RNA double helix can also be included.
  • a plasmid vector containing a T3 promoter sequence was produced.
  • T3 promoter sequence a DNA produced by the use of PCR was used with a view to removing the complexity of the 3-D structure of DNA.
  • the signals shown by schematization of the movement of genes predicted by the T3 promoter sequences and by the method of the present invention for analyses of DNA were as given in Fig. 11 A.
  • the T3 promoter sequence (20bp) shown in Fig. 11 A was input in a microprocessor, and after the following in vitro transcription system was adjusted so that it might come to the center of the regulation device, a cyclic magnetic field specific to said sequence was created. After irradiating a solution of plasmid vector containing a T3 promoter, T3 RNA polymerase, and other buffer solutions with the thus created cyclic magnetic field, the level of the in vitro transcription was estimated by measurement of the quantity of RNA transcribed from the synthesized T3 promoter.
  • the transcription system employed in this example was the Roche's in vitro transcription system (Catalog No. 999-644, Mannheim, Germany).
  • the apparatus of the present invention for regulation of the expression of genes, used in this example, was one, in which electromagnets with 10 poles were radially and horizontally arranged.
  • the cycle of magnetic irradiation was 0.2Hz, the time for irradiation 20 or 40 minutes, the intensity of magnetic field of 20 gausses, and the temperature, room temperature. Meanwhile, the level of in vitro transcription in plasmid containing said T3 promoter, but not irradiated with cyclic magnetic fields, as a control, was measured.
  • the level of in vitro transcription after irradiation of a sequence complementary to a T3 promoter with a cyclic magnetic field specific to that sequence was measured.
  • the cyclic magnetic field specific to the DNA sequence was generated by using the method of distinguishing sequences by the length of time. That is, for A or T power of 1-2 pulses was input a time, and for G or C power of 2-4 pulses, while T and C were distinguished by reversion of the driving power signals from A and G, respectively.
  • the results of this example are given in Fig. 11B. In Fig.
  • A shows the quantity of the RNA expressed in the T3 promoter sequence at the induction by the apparatus of the present invention
  • B shows the quantity of the RNA expressed in the complementary sequence of the T3 promoter at the induction by the apparatus of the present invention
  • Cl and C2 indicate the controls, viz. those that were not irradiated with cyclic magnetic fields, and GRl and GR2 those induced by the apparatus of the present invention while Cl and GRl show the results of the reaction under the same condition for 20 minutes, and C2 and GR2, for 40 minutes.
  • Fig. 11C shows the absorbance patterns each lane at the EtBr absorbance wavelength after EtBr was treated by the use of a densitometer. As will be seen in Fig.
  • GRl shows far more RNA than Cl does, and GR2 far more than C2 does, and all this means that, when the T3 promoter base sequence is affected by the apparatus of the present invention, the RNA product in a unit time greatly increases than in the controls.
  • the T3 promoter base sequence is irradiated with a cyclic magnetic field specific to it, it was found that the amount of the in vitro transcription remained similar or rather decreased, as will be seen in B of Fig. 11B.
  • the base sequence (lObp) in the area was input in the microprocessor of the present apparatus, and a cyclic electromagnetic field specific thereto was generated for irradiation of the mice therewith.
  • the regulation device used in this example was one, in which magnets with 10 poles were each arranged vertically, and again radially.
  • the frequency of the cyclic magnetic field was 0.2Hz, and the intensity of the magnetic field, 10 gausses.
  • a method was adopted, in which the cyclic magnetic field specific to the DNA sequence distinguished A, T, G or C by the length of time and the reversion of the driving power signals. That is, for A or T power with 1-2 pulses a time was input, and for G or C, power with 2-4 pulses, while T and C were distinguished respectively from A and G by reversion of the driving power signals.
  • mice were irradiated with cyclic magnetic fields having the aforesaid characteristics for three hours, and they were given a rest for two hours, and then their blood serum was separated.
  • a conventional ELISA was conducted with the separated serum.
  • the light absorbance at 410nm (OD 410 ) was measured. The value of this absorbance was proportionate to the quantity of the alkaline phosphatase. The results of the experiment are given in Fig. 12.
  • Fig. 12 1 indicates the mice of the control group, which were not irradiated with magnetic fields; 2 illustrates the data on the analyses of the alkaline phosphatase obtained from the mice irradiated with the magnetic field under the aforesaid conditions.
  • Fig. 12 it could be seen that the density of the alkaline phosphatase in the serum separated from the mice which were irradiated with the particular cyclic magnetic field specific to the sequence in the promoter region, grew larger than in the control group. Even though studies should yet be made further of the influences of their tension like the shock, uneasiness, etc.

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Abstract

L'invention concerne un procédé permettant de réguler l'expression génique en utilisant un champ magnétique cyclique ou, plus précisément, un appareil permettant de réguler l'expression génique en utilisant un champ magnétique cyclique et le procédé associé, qui consiste à déterminer la séquence d'une séquence de base ADN ou ARN d'activité biochimique obtenue par analyse qui prédit le chemin du mouvement des électrons libres induits entre les paires de base dans la liaison hydrogène d'une hélice double d'ADN ou d'ARN dans des conditions naturelles ou en présence d'un champ électromagnétique, ou par interrogation de base de données d'ADN ou d'ARN; et à irradier le champ électromagnétique cyclique en fonction de ladite séquence. L'invention concerne également un appareil permettant de réguler l'expression génique par un champ magnétique cyclique et, plus précisément, à un appareil comprenant un microprocesseur qui reçoit des signaux de séquence de base déterminés comme étant biologiquement actifs par rapport au gène à exprimer et qui les sort conformément à la séquence de base; un pilote qui reçoit les signaux de séquence de base provenant du microprocesseur et qui sort des signaux de puissance d'attaque pulsés conformément aux signaux reçus; et un réacteur de champ magnétique comportant des aimants produit un champ magnétique cyclique conformément aux signaux de puissance d'attaque reçus du pilote grâce à l'utilisation des aimants.
PCT/KR2001/000896 2000-10-12 2001-05-28 Dispositif et procede de regulation de l'expression genique a champ electromagnetique cyclique WO2002031103A1 (fr)

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WO2013012276A2 (fr) * 2011-07-19 2013-01-24 주식회사 진바이오시스 Régulateur génique d'une réaction magnétique de l'hydrogène et procédé de régulation génique
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