hapr 3 Result ＆ Discussion
C. elegans behavior
5.1 Real-time PCR system
3.5.1.1 Real –time PCR procedure
Fig 33. Real –time PCR procedure
3.5.2 Gene expression
We use real-time PCR to prove gene expression. The following axes of ordinates of picture (Y axle) Show the disparity that the gene expression. Disparity that the gene expression is a sub number of 2 Gene expression = 2n
Now we give an example in 3.5.3 Fist real-time PCR test(flow) For example1, gene number 31 Y axle is similar -3
Gene expression = 2-3 = 8-1
We can say in this gene number 31 gene expression the experiment is 8 times lower than the control.
So gene number 31 Gene expression was reduced in the mechanism of testing at this moment. And it has reduced by 8 times
For example2, gene number 75 Y axle is similar 14 Gene expression = 214 = 16384
We can say in this gene number 75 gene expresssion in the experiment is 16384 times higher than thecontrol. So gene number 75 Gene expression increased in the mechanism of testing at this moment. And it has increased 16384 times.
3.5.3 Fist time real-time PCR test
control N2
experiment N2+Fe3O4+MF
The axes of ordinates of chart(Y axle) are t △gene expression(the experiment－the control) The axes of horizontal of chart(X axle) are the gene name serial numbers
The result is not believable, because it is only work one time.
The positive number expresses the experiment group ＞ the control.
The negative number expresses the experiment group ＜ the control (data base on Fig 34)
Fig 34. Fist time real-time PCR data test
3.5.4 Real-time PCR data believable The result is believable. It is three time replay.
Real-time PCR is very sensitive, so we must utilize knowledge, experience and effective tool to get rid of useless data. A simple method is to see whether PCR Amp/Cycle and Melt Curve accord with expectancy.
Prove by following 2 Graph. We adopt just with PCR curve intact and sharp Melt Curve. Because data is numerous, we got rid of no influencing and data disorderly. The following data is all covering triply, the credibility is very high
PCR Amp/Cycle Graph for SYBR-490
Fig 35. PCR Amp/Cycle Graph for SYBR-490
Melt Curve Graph for SYBR-490
Fig 36. Melt Curve Graph for SYBR-490
3.5.4.1 Ced family gene expression
Fig 37.Ced real-time PCR data to gene expression
Ceds are very famous for apoptosis research gene . The control is normal N2
The experiment 1 is N2 +Fe3O4
The experiment 2 is N2 +Fe3O4+mabnetic field (MF)
We can observe that all gene expression increase with magnetic field obviously. Though add Fe3O4 to gene expression has influence, but we can confirm the magnetic field has really influenced gene expression even more.
3.5.4.2 Other apoptosis related gene expression
Fig 38.Other apoptosis related gene real-time PCR data to gene expression
The control is normal N2 The experiment 1 is N2 +Fe3O4
The experiment 2 is N2 +Fe3O4+mabnetic field (MF) These apoptosis related genes are all very famous.
Take abl-1 as an example:
A very obvious one that adds Fe3O4 to C.elegans is not affected. But gene expression is very obvious under magnetic field function. Magnetic field function disparity is reached 25 = 32 times.
3.5.4. Some cancer related gene expression
Fig 39.Some cancer related gene real-time PCR data to gene expression
The control is normal N2 The experiment 1 is N2 +Fe3O4
The experiment 2 is N2 +Fe3O4+mabnetic field (MF
We are very sorry about that it is not easy to look over t the chart small very much.
But we can still perceive the magnetic field has really influenced gene expression.
3.6 C.elegans GFP marked
Fig 40. GFP mark signal
From CGC mutant C.elegans, some strain has GFP mark; .liko cbp-1.Cbp-1 has GFP mark in pharyngeal. We culture cbp-1 C.elegans with the magnetic field 96 hr (right) and with no magnetic environment (left).
Fig 41.GFP signal decrease with magnetic field
We find an interesting thing that pharyngeal GFP signal will be reduced acting on continuously with magnetic field. Though CBP-1 can change with the magnetic field, pharyngeal GFP signal is not reporter gene GFP signal. Although the cbp-1 gene expression decrease with magnetic field in our experiment, it can't be proved while making people interesting like this.
3.7 C.elegans mutant behavior ＆ real-time PCR data
Fe3O4 ultra paramagnetic nanoparticles influence gene expression and strengthen the impact on gene expression of the magnetic field. But this ultra paramagnetic nanoparticles has spontaneous magnetic field. In order to distinguish the spontaneous magnetic field and the magnetism field influence. We join 2 kinds of nonpoisonous nanoparticles-Fe2O3, TiO2...Fe2O3andTiO2.diameter are (40nm) slightly biger than Fe3O4 (10nm).
Fe2O3 is compared with Fe4O3; they are all oxides of the iron. The difference is the ultra paramagnetic in Fe3O4 has spontaneous magnetic fields. The same one they are attracted by magnetic field. : They all move with the direction of the magnetic field. Join Fe2O3 in order to prove what is influenced is in the experiment. It was not only a spontaneous magnetic field that was influenced, the more important thing environment magnetic field.
TiO2 is compared with Fe4O3. They are all nonpoisonous nanoparticles. Fe3O4 ultra paramagnetic nanoparticles influence gene expression and strengthen the impact on gene expression of the magnetic field. Nanoparticle influence gene expression or environment magnetic field. This is important thing. TiO2 is useful for riding of the influence of the iron oxide and distinguishing nanoparticles influences.
We request strains from CGC
C.elegans mutant gene CGC strain C.elegans mutant gene CGC strain
unc-30 EW45 unc-119 XA3504
Table 4 . Request strains from CGC
Now we use 3 new mutant strains, ced-3 MT1522, ced-6 MT4970, cbp-1 VC1006 to prove our result.Ced-3 and ced-6 are apoptosis related gene. Cbp-1 is cancer related gene. They are all deficient mutant.Ced-3 and ced-6 is in the same pathway in apoptosis. We find apoptosis deficient mutant is insensitive to magnetic field.
Mutant location genomic environs
Ced-3 MT1522
Ced-6 MT4970
Cbp-1 VC1006
Fig 42 Ced-3, ced-6, cbp-1 mutant location genomic environs
3.7.1 Ced-3 MT1522
3.7.1.1 Ced-3 mutant behavior
Fig 43. Ced-3 mutant behavior with magnetic field
The above is taken a sample at random by 25, ρ＜0.05.Recording mode is as same as before utilizing. We compare normal N2 with ced-3 mutant C.elegans. The result is that ced-3 mutant C.elegans is not sensitive to the magnetic field.
3.7.1.2 Ced-3 mutant real-time PCR data
Fig 44. Ced-3 mutant real-time PCR data to gene expression
The type adds the kind of nanoparticles above the chart (bar1 has no nanoparticle)
It is three time replay. Different display ways before following. : Question that we melt simply.
Whether only the magnetic field will have changes to ced-3 mutant C.elegans. The result is excited, not adding any nanoparticle, (bar1)
Gene expression with magnetic field increase more than 5 times of Gene expression without magnetic field. This result expresses magnetic field and ced-3 positive correlation.Ced-3 is important apoptosis pathway. We can suppose boldly the magnetic field correlate with apoptosis.
The magnetic field causes apoptosis.
The magnetic fields still change gene expression and it is positive correlated. Though seem to add nanoparticles (Fe3O4 Fe2O3 TiO2) reduce the difference. We do not care about this. Because on generally speaking, it contain inside great and complicated problem that are not solved.
3.7.2 Ced-6 MT4970
3.7.2.1 Ced-6 mutant behavior
Fig 45. Ced-6 mutant behavior with magnetic field
The above is taken a sample at random by 25, ρ＜0.05 Recording mode is as same as before utilizing.
we compare normal N2 with ced-6 mutant C.elegans.
Two of their results are similar. The possible reason is as follows 1. Ced-6 mutant damage is not serious
2. Gene relevant degree is not enough 3. Gene pathway not related directly
Behavior datas show ced-6 mutant and magnetic field function have no relation between with themselves. The result accords with the expectancy of apoptosis pathway
No matter how, we can still affirm there is an influence in the magnetic field
3.7.2.2 Ced-6 mutant real-time data
Fig 46. Ced-6 mutant real-time PCR data to gene expression
If C.elegans gene related to phagocytosis mutant in the body, liko ced-6, may escape death.
Except that the passive role with death cell's skeleton of a corpse of phagocyting and is not merely acted and moved around the cell. Even cause the initiative assailant of apoptosis [31].
Ced-3 and ced-6 are not in same pathway , although they are apoptosis related. The result shows the lower gene expression with magnetic field function.
Fig 47.ced-6 apoptosis related pathway
3.7.3 Cbp-1 VC1006
3.7.2.1 Cbp-1 mutant behavior
Fig 48. Cbp-1 mutant behavior with magnetic field
The above is taken a sample at random by 25, ρ＜0.05.
Cbp-1 mutant is compared with N2.
We look cbp-1 mutant C.elegans is more sensitive to the magnetic field instinctively. This kind of method instinctively lacks the scientific idea.
N2 speed under magnetic field function reduces almost 3 mm/min. (N2 vs. N2+MF from 7 to 4) Cbp-1 mutant speed under magnetic field function reduces almost 3 mm/min. (cbp-1 vs. cbp-1+MF from 5.5 to 2.5).
Their disparity is all 3. We can not put the final conclusion that cbp-1 mutant C.elegans is more sensitive to the magnetic field instinctively.
3.7.2.2 Cbp-1 mutant real-time data
Fig 49. Cbp-1 mutant real-time PCR data to gene expression
Over 80% of the cancers known produce, because p53 is lost badly at present.
P53 is important, because it has 3 functions:
1. Order cells not to grow again, is engaged in the work of mending damaged DNA attentively (growth arrest).
2. When being unable to mend, order cells to commit suicide.
3. Suppress the blood vessel hyperplasia (anti-angiogensis), Supply the cancer cell with a nutrient, and make the cancer cell unable to grow up, shift.
Produce the cancer, but destroy p53 .Celegans cbp-1 is similar to human P300/CBP
P300/CBP is activating of p53, by combining with p53 N end, and 382 acetylating lysine, increase p53 activate the ability that the gene expression. [32] Cbp-1 gene expression accords with expectancy. Cbp-1 gene expression is cancer negative related.The result infers the magnetic field correlate with cancer genes.
Chapter 4 Conclusion.
Our initial discovery is that we find normal C.elegans N2 behavior is unusual under magnetic field function. Later we combine with super paramagnetic nanoparticles and magnetic field in our experiment. The result is more remarkable. We suppose the magnetic field also influence other physiological mechanisms besides behavior. In numerous real-time PCR data are analyzed, we find out the gene correlated with magnetic field thoroughly. Biological mechanism and gene expression pathway to C.elegans about the magnetic field are we design more than one hundred primer pairs in order to analyze gene expression. Certainly there are a lot of topics worthy to be deep we have not finished yet. By our present experimental result, we think the magnetic field causes the influence on the gene and behavior on the living beings.
1. The magnetic field influences C.elegans behavior.
C.elegans creeps slowly under magnetic field function. Noted down and all shown the magnetic field influence behavior by statistical graph or video. Mostly correlate with nervous system about research of C.elegans behavior. Nervous system relevant pathological changes have many such as Alzheimer's disease it is a question that is worth going into seriously.
2. Magnetic field is induction of apoptosis
Showing from real-time PCR data, there is a lot of apoptosis, cancer, oxidation all correlate with magnetic field function. In order to confirm the result even more. We have concentrated resources on finding out ced-3 and ced-6 gene that apoptosis are correlated with.Cbp-1 gene is correlated with cancer. Because of cbp-1 mutant behavior data, the magnetic field and cancer direct relation that we can not come to a conclusion.
3. Apoptosis deficient mutant is insensitive to magnetic field
Ced-3 is important key of apoptosis pathway. Ced-3 begins function expression and then the living cell switch a succession of apoptosis mechanisms. We use mutant C.elegans to prove apoptosis deficient mutant is insensitive to magnetic field. Reference [33] echoes with our result.
(Fig50) .They also use mutant ced-3 and ced-6. Ced-3 alone mutant is not apt apoptosis. Ced-6 mutant is not relevant directly to apoptosis, but ced-3 and ced-6 pairs of mutant more difficult to apoptosis.
Ced-3 mutant is not sensitive to the magnetic field (our experiment)
Ced-6 is pathways to regulate cell corpse engulfment.Ced-6 does not participate in apoptosis directly [33]. Our results reflect by ced-6 behavior data proves ced-6 single mutant not to be obvious to the magnetic field influence. Certainly the result of ced-3 and ced-6 accord with expectancy .The magnetic field relates to apoptosis directly. We confirm the magnetic field cause apoptosis to the living beings.
Fig 50 Reference [33] some data
Future prospect
Relevant researches about magnetic field influence living beings are worth our further investigating very much. We can find out the physiological factor that the magnetic field influences to C.elegans in succession in the future. We can make C.elegans life span. Still we prove other gene magnetic fields are related more accurately. We have proved 3 genes after all.
In addition, the proteins that the magnetic field related will be proved in the direction of our future.
Chapter 5 Reference
[1].. Extremely low frequency electromagnetic .elds (EMF) and brain cancer in adults and children: Review and comment Environmental Health Perspectives, Vol. 110, Supplement 4.
(Aug., 2002), pp. 613-618.
[2] World Health Organization, International Agency for Research on Cancer. Volume 80:
Non-ionizing radiation, Part 1, Static and extremely low-frequency (ELF) electric and magnetic fields. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. 2002: Lyon, France.
[3] Wertheimer N, Leeper E. Electrical wiring configurations and childhood cancer.
American Journal of Epidemiology 1979; 109(3): 273–284.
[4]. Kliukiene J, Tynes T, Andersen A. Residential and occupational exposures to 50-Hz magnetic fields and breast cancer in women: A population-based study. American
Journal of Epidemiology 2004; 159(9): 852–861.
[5] Zhu K, Hunter S, Payne-Wilks K, et al. Use of electric bedding devices and risk of breast cancer in African-American women. American Journal of Epidemiology 2003; 158:
798–806.
[6] Tynes T, Haldorsen T. Residential and occupational exposure to 50 Hz magnetic fields and hematological cancers in Norway. Cancer Causes & Control 2003; 14: 715–720.
[7]. Labreche F, Goldberg MS, Valois M-F, et al. Occupational exposures to extremely low frequency magnetic fields and postmenopausal breast cancer. American Journal of
Industrial Medicine 2003; 44: 643–652.
[8]. Willett E, McKinney PA, Fear NT, et al. Occupational exposure to electromagnetic fields and acute leukaemia: Analysis of a case-control study. Occupational and
Environmental Medicine 2003; 60: 577–583.
[9] Biological responses in Caenorhabditis elegans to high magnetic fields.
Experientia. 1995 Mar 15;51(3):284-8.
[10] Transgenic nematodes as biomonitors of microwave in ducedstress. 1998 Mar 13;399(1):5564. Daniells C, Duce I, Thomas D, Sewell P, Tattersall J, de Pomerai D.
Department of Life Science, University of Nottingham, University Park, UK.
[11] Electromagnetic Fields Enhance the Stress Response at Elevated Temperatures in the NematodeCaenorhabditis elegans .Bioelectromagnetics 21:100^106 (2000)
Bernd Junkersdorf,1 Hartmut Bauer,2 and Herwig O. Gutzeit1* 1Institut fÏr
Elektroenergieversorgung,Technische UniversitÌt Dresden, Dresden, Germany
[12] .Exposure of Caenorhabditis elegans to extremely low frequency high magnetic fields induces stress responses. Bioelectromagnetics. 2001 Jul;22(5):3339. Miyakawa T, Yamada S, Harada S, Ishimori T, Yamamoto H, Hosono R.
Laboratory of Magnetic Field
[13]. Biogenic magnetite in the nematode caenorhabditis elegans.Proc Biol Sci. 2004 Dec 7;271 Suppl 6:S436-9.
[14]. 50–60 Hz ELECTRIC AND MAGNETIC FIELD EFFECTS ON COGNITIVE FUNCTION IN HUMANS: A REVIEW Radiation Protection Dosimetry Vol. 106, No 4, pp.
333–340 (2003)
[15] Review Article Low-Level Exposure to Radiofrequency Electromagnetic Fields:
Health Effects and Research Needs .Bioelectromagnetics 19:1–19 (1998)
[16.] Exposure to magnetic field (5 mT at 60 Hz) does not affect cell growth and c-myc gene expressi J.radiat.res.,37,185-191(1996)
[17] Magnetic Fields and Cancer: Animal and Cellular Evidence-an Overview Bo Holmberg .National Institute of Occupational Health, Solna, Sweden
[18] Magnetic field stress induces expression hsp 70 Cell Stress ＆ Chaperons (1998)3-{2},79-88
[19] High magnetic field induced changes of gene expression in arabidopsis Anna-Lisa Paul1, Robert J Ferl1 and Mark W Meisel*2.BioMagnetic Research and Technology 2006, 4:7 [20] A Magnetic Field-Responsive Domain in the Human HSP70 Promoter.Journal of Cellular Biochemistry 75:170–176 (1999) .H. Lin,1 M. Blank,2 and R. Goodman1*
[21] Magnetite in Human Tissues: A Mechanism for the Biological Effects of Weak ELF Magnetic Fields- Bioelectromagnetics Supplement 1 :101-113 (1992)
Joseph L. Kirschvink, Atsuko Kobayashi-Kirschvink, Juan C. Diaz-Ricci, and Steven J.
Kirschvink
[22 ] Toxicity of iron oxides and metabolites of benzo[a]pyrene alone or in combination in cells culture and identification by laser microprobe mass spectrometry. Cent Eur J Public Health. 1996;4 Suppl:58-9.
[23] Toxicity of metal oxide nanoparticles in mammalian cells.1: J Environ Sci Health A Tox Hazard Subst Environ Eng 2006;41(12):2699-711.
[24] Detection of specific polymerase chain reaction product by utilizing the 5’–3’
exonuclease activity of Thermus aquaticus DNA polymerase Holland PM, Abramson RD,Watson R, Gelfand DH.. Proc Natl Acad Sci USA 1991; 88:7276–7280.
[25] Simultaneous amplification and detection of specific DNA sequences.Higuchi R, Dollinger G, Walsh PS, Griffith R. Biotechnology 1992;10:413–417.
[26] PCR analysis: real-time monitoring of DNA amplification reactions.Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic Biotechnology 1993;11:1026–1030.
[27] The polymerase chain reaction.J Clin Pathol. 1989 Jul;42(7):673-6.
[28] Breaking the Mitochondrial Barrie 1. Martinou J.C. and Green D.R. r. Nat. Rev. Mol.
Cell. Biol. (2001); Vol. 1: pp.63-67.
[29] Disruption of p53 in J. Clin. Bunz F., Hwang P.M., et. al.. Invest. (1999); Vol. 104:
pp.263-69.
[30] An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis.Nat Genet. 2005 Jan;37(1):7-8. Sweet-Cordero A, Mukherjee S, Subramanian A, You H, Roix JJ, Ladd-Acosta C, Mesirov J, Golub TR, Jacks T.
[31] Reddien, P. W., Cameron, S. & Horvitz, H. R. Phagocytosis promotes programmed cell death in C. elegans. Nature 412: 198-202 (2001).
[32] A Gain-of-Function Allele of cbp-1, the Caenorhabditis elegans Ortholog of the Mammalian CBP/p300 Gene, Causes an Increase in Histone Acetyltransferase Activity and Antagonism of Activated Ras. MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p.
9427–9434.
[33] Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans Nature. 2001 Jul 12;412(6843):133-5 Hoeppner DJ, Hengartner MO, Schnabel R.