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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2093 - 1/6
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`

`
`~
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`
`NAIIIIIE
`
`MACMILLAN JOURNALS LTD
`
`No. 5414
`
`FRIDAY AUGUST 3 1973
`
`Vol. 244
`
`L O N D O N
`4 Little Essex Street, London WC2R 3LF. Tel : O1 836 6633 Telex : 262024
`
`W A S H I N G T O N
`
`711 National Press Building, Washington DC 20004. Tel : 202 737-2355 Telex : 64280
`
`CostsandBenefitsofCleanAir
`
`.
`
`.
`
`.
`
`.
`
`.
`
`247
`
`P036’
`
`ow womb
`The Research System as Margaret Thatcher sees it/British
`-
`-
`.
`.
`.
`.
`Qsgogiation/Research Councils/Radrocatrve Waste/
`~
`
`NEW WORLD
`
`248
`
`Fusion Power Changes Gear/NSF/Science Expenditure/
`Executive shume _
`_
`_
`,
`_
`_
`_
`
`250
`
`Environmcnt—Mcrcury in Californian varved marine
`
`sediments—YOUNG,
`JOHNSON (Southern Cali-
`3:322:22?a;:$::,.‘:.::::.::.3
`Geo“)
`.
`gy—Age of Bulawayan stromatol1tes—BOND,
`WILSON and WINNALL (Salisbury, Rhodesia)
`.
`Crystallography—Texture of quartz—WHITE (Imperial
`College, London)
`.
`.
`.
`.
`.
`.
`
`Pollution—Biological method of estimating smoke and
`sulphur dioxide pollution—CREED, LEES (Cardiff)
`and DUCKETT (Bangor)
`.
`.
`.
`.
`.
`
`NEWS AND V|EWS
`Leukaemia Viruses:Human or Feline?/Predation/Neurm
`toxins / Ecology / Polypeptides / Liquids / Oceano—
`graphy/High-energy P11ysics/Low Temperature Phy-
`sics
`.
`.
`.
`.
`.
`.
`.
`.
`.
`
`ARTWS
`Ancient Linkage Groups and Frozen Accidents—OHNO
`(City of Hope Medical Center)
`
`'
`
`.
`
`.
`
`Cosmol0gy—Experimental constraints on future proper-
`ties of the UniVerse—PARTRIDGE (Haverford)
`.
`
`‘
`
`252
`
`259
`
`263
`
`266
`
`Industrial healtl1—Efl‘ect of heat on X-ray diffraction
`§§lt%2n(:;:;be§:):I;PI:g::A:;A)N (Central Elect”
`y
`g
`’
`g C
`'
`'
`'
`'
`Optics—Reduction of lens reflexion by the “moth eye”
`principle—CLAPHAM and HUTLEY (National
`Physical Laboratory, Teddington)
`.
`.
`.
`.
`‘za°°:’:::r
`a
`1 gfe
`National Laboratory) and MCHUCIH (Knolls Atomic
`.
`.
`.
`
`4
`
`.
`
`C
`'
`,
`’
`.
`Power Laboratory, Schenectady)
`
`Biological Sciences
`tRNA—l-lypermethylation in cleaving rabbit embryos—
`MANES and SHARMA (Colorado)
`.
`.
`.
`
`Histamine receptors—Increase on thymus-derived effec-
`tor lymphocytes during the primary immune response
`to
`alloantigcns—PLAUT, LICHTENSTEIN and
`HENNEY (Johns Hopkins and O’Neil1 Memorial
`Research Laboratories, Baltimore)
`.
`.
`.
`
`Notes of articles appearing next week in Nature
`Physical Science and Nature New Biology will be found
`on pages 255 to 257.
`
`(Caminued overleaf
`
`Feline leukaemia virus—Field study on horizontal trans-
`mission—HARDY, OLD (Memorial Sloan-Kettering),
`HESS (Henry Bergh Memorial Hospital, New York),
`ESSEX (Harvard) and COTTER (Angeli Memorial
`Animal Hospital, Boston)
`.
`.
`.
`.
`.
`
`LETTERS TO NATURE
`Physical Sciences
`
`Radio astronomy~—5 GHz emission from Algol—
`POOLEY and RYLE (Cambridge)
`.
`.
`.
`
`Solar system——Noble gases in the Titan atmosphere-
`CESS and OWEN (Stony Brook)
`.
`.
`.
`.
`
`270
`
`272
`
`NATURE is published weekly by Macmillan Journals Ltd., 4 Little Essex Street, London WC2R 3LF. Direct all manuscripts, subscription
`orders and enquiries, and correspondence to: Nature, 711 National Press Building, Washington DC 20004. Publication address, The William
`Byrd Press |nc., 2901 Byrdhill Road, Richmond, Va. 23228. Annual subscription in US: Nature E20; Nature and Nature Physical Science
`£33; Nature and Nature New Biology £33; all three editions E44; Index £1.50.
`Second-class postage paid at Richmond, Va.
`Printed in USA.
`POSTMASTER: Please send Form 3579 to: Nature, 711 National Press Building, Washington DC 20004.
`
`CFAD V. Anacor, |PR2015-01776 ANACOR EX. 2093 - 2/6
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2093 - 2/6
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`

`
`Histamine receptorsalnhibition of histamine release is
`controlled by an H2 rcccptor—LICHTENSTEIN and
`GILLESPIE (Johns Hopkins)
`.
`.
`.
`.
`
`Platelets—Inhibition and reversal ofplatelet aggregation
`by cytochalasin B or colcemid—KAY and FUDEN-
`BERG (University of California School of Medicine,
`San Francisco)
`.
`.
`.
`.
`.
`.
`.
`
`investigated by somatic
`Cystinotic cells—Properties
`cell
`hybridization—SCHNElDER,
`FRANCKE,
`HAMMOND, PELLETT and BECKER (La Jolla)
`.
`
`Griseofulvin—Infiuence on fungal mitosis—GULL and
`TRINCI (Queen Elizabeth College, London)
`.
`.
`
`—Does not afl'ect microtubule assembly-
`GRISI-IAM, WILSON, and BENSCI-I (Stanford)
`.
`
`Insect photoperiodism—Responses, to daylength during
`diapause—TAUBER and TAUBER (Cornell) .
`.
`Surface membrane charges —— Changes in distribution of
`electric charges in the surface membrane of rat ascites
`hepatocarcinoma cells induced by con A——YAMADA
`and YAMADA (National Cancer‘ Centre Research
`Institute, Tokyo) .
`.
`.
`.
`.
`.
`.
`
`Mitotic synchrony—Inducti0n by intermittent hyper-
`thermia in Ehrlich carcinoma grown in viva-
`SAPOZINK, DESC!-INER and HAHN (Memorial
`Sloan-Kettering)
`.
`.
`.
`.
`.
`.
`.
`
`Myleran—Influence on two normal haemopoietic pro-
`genitor cell lines—BLACKETT and MILLARD (ICR,
`Sutton)
`.
`.
`.
`.
`.
`.
`.
`.
`
`Immunosuppression—Efl‘ect of Toxoplasma gondii on
`mouse thymus—HULDT, GARD and OLOVSON
`(National Bacteriological Laboratory, Stockholm)
`.
`
`Osteoarthritis—Possible development of early osteo-
`arthritic fibrillation caused by cartilage fatigue-
`WEIGHTMAN, FREEMAN and SWANSON (Im-
`perial College, London)
`.
`.
`.
`.
`.
`
`Fungal genetics~Suppression of monokaryotic fruiting
`in Basidiomycetes by incompatibility factors—ESSER
`and STAHL (Ruhr-Universitat, Bochum)
`.
`.
`
`Nematotles—Antigens in common with host plants-
`MCCLURE, MISAGHI and NIGH (Arizona)
`.
`
`Page
`
`287
`
`288
`
`289
`
`292
`
`294
`
`296
`
`297
`
`299
`
`300
`
`301
`
`303
`
`304
`
`306
`
`Phyt0plankton—Rem0te sensing from space aids produc-
`tivity in pelagic fisheries—YENTSCIl (Massachusetts)
`Latimeria retina—Possible discontinuous rod outer
`segment f0rmation—LOC1(ET (Institute of Ophthal-
`mology, London)
`.
`.
`.
`.
`.
`
`Sauropoda—Haemodynamics of Aptosaurus and Bru-
`c/u'osam'us—HOHNKE (Pfizer Central Research,
`Groton)
`.
`.
`.
`.
`.
`.
`.
`
`Plant ecology—Diversity of herbaceous vegetation on
`fertile soil—NEWMAN (Bristol)
`.
`.
`.
`
`.
`—Reply—GRIME (Sheffield)
`for
`Chromosomes—Corrected chromosome number
`Sparfina in Ireland—BOYLE (University College,
`Dublin)
`.
`.
`.
`.
`.
`.
`.
`
`Rhodesian man — Geological
`(Chicago)
`.
`.
`.
`
`.
`
`antiquity —- KLEIN
`.
`.
`.
`
`Ramapithecus—ReC0nslructi0n of dental arcades-
`WALKER and ANDREWS (Nairobi)
`.
`.
`
`Child psychc‘_0gy—Inferences and memory—YOUNISS
`and FURTH (Catholic University ofAmerica) .
`
`—Reply—BRYANT (Oxford)
`
`.
`
`General
`
`Consumer purchasing—Frequency distribution for differ-
`ent brands;GOODHARDT and CHATFIELD
`(London and Bath)
`.
`.
`.
`.
`.
`
`BOOK REVIEWS
`
`Agrarian Conditions in Northern India, Elizabeth Whit-
`combe (GORDON CONWAY)
`.
`.
`.
`
`Developmental Physiology and Aging, P. S. Timiras
`(A. COMFORT)
`.
`.
`.
`.
`.
`
`Genetics: a Basic Guide, I. J. Pedder and E. G. Wynne
`(R. A. WOODS)
`.
`.
`.
`.
`.
`.
`Structured Programming, 0. J. Dahl, E. W. Dijkstra and
`C. A. R. Hoare (NOEL FALCONER)
`.
`.
`
`Obituary—-Dr E. Bretscher, CBE
`
`.
`
`.
`
`.
`
`Announcements—University News, Miscellaneous
`
` *—j
`
`Next Monday’s issue will include
`Relativity—f gravity and gravitational
`collapse——ISHAM,
`SALAM (Imperial College, London) and STRATHDEE
`(International Centre for Theoretical Physics, Trieste)
`
`Pulsars —Thirteen new ones discovered—DAVIES, LYNE and
`SEIRADAKIS (Jodrell Bank)
`
`Cosmic rays—Exp1anation of “step” in Leeds spectrum-
`O’BR1EN (US Atomic Energy Commission, New York)
`
`Cos1nology—Ca11 spin avert singularities?—STEWART and
`HAJICEK (Max-Planck-Institut, Munchen)
`
`Next Wednesday’s issue will include
`5-Hydroxytryptamine—Excitatory and inhibitory responses
`resulting from a decrease in membrane permeability-
`PAUPARDIN-TRITSCH and GERSCHENFELD (Ecole
`Normale Superieure, Paris)
`
`Contact sensitivity——Anatomical location of cells that mediate
`contact sensitivity in the lymph nodes and bone marrow-
`ASHERSON and ZEMBALA (Clinical Research Centre,
`Harrow)
`
`Diabetes—Changes in aldolase isoenzymes of adipose tissue
`induced by diabetes and ATP——BEITNER and LAHAT
`(Bar-Ilan)
`
`RNA—Analysis of axoplasmic RNA from invertebrate giant
`axons—LASEK, DABROWSKI
`and NORDLANDER
`(Case Western Reserve and Woods Hole)
`
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`

`
`NATURE VOL. 244 AUGUST 3 1973
`
`1° Littlefield, J. W., Science, 145, 709 (1964).
`1‘ Gartler, S. M., Nat. Cancer Inst. Monogr., 26, 167 (1967).
`1’ Steele, M. W., Bioclmm. Genet., 4, 25 (1970).
`1“ Yoshida, A., Watanabe, S., and Gartler, S. M., Biacltem. Genet., 5,
`533 (1971).
`1" Miller, 0. J., Miller, D. A., Allderdice, P. W., Dev, V. G., and
`Grewal, M. S., Cytogenetics, 10, 338 (1971)._
`1‘ Francke, U., Hammond, D. S., and Schneider, J. A., Chromosoma
`41, 111 (1973).
`1'’ Matsuya, Y., and Green, H., Science, 163, 697 (1969).
`17 Silagi, S., Darlington, G., and Bruce, S. A., Proc. US Nat. Acad.
`Sci., 62, 1085 (1969).
`1‘ Hors-Cayla, M. C., Lyon, G., Heuertz, S., Maroteaux, P., and
`Frézal, J., Fourtli Intern. Cangr. Human Genet. (abstract, 1971).
`1” Schneider, J. A., and Weiss, M. C., Proc. US Nat. Acad. Sci., 68,
`127 (1971).
`7" Caspersson, T., Zech, L., Johansson, C., and Modest, E. J.,
`Chromosoma, 30, 215 (1970).
`“1 Scabright, M., Lancet, ii, 971 (1971).
`” Pollock, M. E., Kenny, G. E., and Syverton, J. T., Proc. Soc. Exp.
`Biol. l\lea'., 105, 10 (1960).
`*3 Hayflick, L., Texas‘ Rep. Biol. Med., 23, 285 (1965).
`“ MacPherson, 1., J. Cell Sci., 1, 145 (1966).
`‘*5 Boyer, S. H., Fainer, D. C., and Naughton, M. A., Science, 140,
`1228 (1963).
`2“ Ruddle, F. H., and Nichols, E. A., In Vitro, 7, 120 (1971).
`
`Griseofulvin inhibits Fungal Mitosis
`GRISEOFULVIN is an antifungal antibiotic widely used for the
`treatment of human and animal dermatophytic infections. The
`in vivo antifungal activity of griseofulvin is a reflexion of its
`peculiar pharmacological properties for, after oral admini-
`stration,
`it
`is specifically localized and concentrated in the
`keratinized cells of the skin, hair and nails. The invasive growth
`of dermatophytes parasitizing these tissues is inhibited and the
`infected tissue is removed by desquamation and the growth of
`new epidermal layers. Here we report the effects of griseofulvin
`on fungal mitosis.
`Basidiobolus ranarum was used. This has one very large
`nucleus (25 um long) in each hyphal compartment (Fig. 1). After
`mitosis, the daughter nuclei move rapidly apart and the com-
`partment is divided by the centripetal growth of a septum which
`has no central pore. Thus, each hyphal compartment may be
`regarded as a single uninucleate cell. B. ranarum was grown at
`30° C in Petri dishes containing 20 ml of a malt extract, yeast
`extract, glucose, peptone agar medium. The peripheral hyphae
`
`Table 2 Free Cystine Content of Parent and Hybrid Clones
`
`“Parent" cells
`
`Cystinotic fibroblasts
`
`Hybrid cells
`DC 11
`DC 14
`DC 20
`DC 22
`DC 23
`DC 24
`DC 26
`DC 30
`DC 31
`DC 33
`DC 34
`DC 35A
`DC 37
`
`(nmol 1/2 cystine mg“ protein)
`0.1 1
`
`6.78
`6.05
`
`0.02
`0.04
`<0.01
`0.04
`0.08
`0.03
`0.0]
`0.01
`0.11
`0.06
`0.03
`0.09
`<0.0l
`
`Mean
`
`0.04
`
`Free cystine was measured as previously described‘.
`
`The high cystine content of C-1 indicates that the low cystine
`content in the hybrid clones did not result from these procedures.
`Interpretation of these data is difficult. The question to be
`answered is why none of the hybrid clones was similar to
`heterozygote cells in its cystine content. The heterozygote cell
`receives one normal and one abnormal gene and has a cystine
`content five times greater than normal. The hybrid cell receives
`two abnormal genes and at least two normal genes and has a
`normal content of free cystine. Perhaps two normal genes per
`cell is sulficient even if the cell is very large. Another possibility
`is that the heteroploid (tumour) cell has taken over “control”
`of the hybrid cell and that this “control” includes regulation of
`cystine metabolism.
`One further possibility is that the D98 cell makes a substance
`which can reduce cystine to cysteine in the cystinotic lysosome
`and thus correct the defect. We are presently pursuing this
`possibility.
`We thank Drs Robert Colfman, Robert Hyman, and David
`Hutton who supplied the Sendai virus. This work was supported
`by grants from the National Institutes of Health and the
`American Heart Association. J. A. S. is an Established Investi-
`gator of the American Heart Association.
`JERRY A. SCHNEIDER
`UTA FRANCKE
`DEBORAH S. HAMMOND
`OCEAN L. PELLETT
`FRED L. A. BECKER
`
`Department ofPediatrics,
`University of California,
`San Diego, La Jolla,
`California 92037
`Received April 6, 1973.
`‘ Barski, G., Sorieul, S., and Cornefcrt, F., CR Acatl. Sci., 251,
`1825 (1960).
`2 Ephrussi, B., in Hybridization ofSomatic Cell (Princeton University
`Press, Princeton, New Jersey, 1972).
`3 Schneider, J. A., and Seegmiller, J. E., in The Metabolic Basis of
`Inlzerited Disease (edit. by Stanbury, J. E), 3rd ed., 1581
`(McGraw-Hill, New York, 1972).
`*‘ Schneider, J. A., Bradley, K., and Seegmiller, J. E., Science, 157,
`1321 (1967).
`5' Schneider, J. A., Rosenbloom, F. M., Bradley, K. H., and Seeg-
`miller, J. E., Bioclzem. Biopltys. Res. Commun., 29, 527 (1967).
`‘’ Patrick, A. D., and Lake, B. D., J. Clin. Patliol., 21, 571 (1968).
`7 Schulman, J. D., Bradley, K. H., and Seegmiller, J. E., Science,
`166, 1152 (1969).
`“ Tietze, F., Bradley, K. H., and Schulman, J. D., Pediat. Res., 6,
`
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`
`2.0
`
`50
`
`Griseofulvin (pg ml—l)
`
`400 rControl
`
`200
`
`L:
`E
`ad
`nd
`U
`
`0a
`
`0
`
`I .0
`
`of colonies were stained with 0.2?/,, (w/v) aqueous acridine
`orange for 1 min, washed with water and examined by ultra-
`violet microscopy. The nucleolus fluoresced red and the con-
`densed mitotic chromosomes fluoreseed green. This technique
`facilitated the recognition of the various stages in mitosis.
`A large proportion of hyphae growing on media containing
`5 to 10 ug ml" griseofulvin had compartments which contained
`more than one nucleus (Figs 2 and 3). Of these multinucleate
`compartments 88 % were binucleate, 9‘?/,, were trinucleate and
`3 ‘X, were quadrinucleate. The multinucleate condition of many
`griseofulvin treated hyphae was clearly seen after acridine
`staining (Fig. 3). In these multinucleate hyphal compartments
`the nuclear membrane of each nucleus was apparent, showing
`that each nucleus was physically isolated from the other. even
`though they \ve1'e very close together. Control hyphal compart-
`ments with two nuclei were scen only very rarely, and if such
`
`
`
`Griseofulvin (ug ml" I)
`
`Mitotic index
`
`
`
`Fig. 4 The effect of griseofulvin on the colony radial growth
`rate (CRGR) and mitotic index of Basidiobolus. For growth rate
`experiments
`increasing concentrations of griseofulvin were
`incorporated into solid medium and the radial growth rate of
`colonies measured as previously reportedz. Mitotic index counts
`were performed on parallel colonies growing on medium
`containing griseofulvin. Peripheral mycelium was stained in
`acridine orange and viewed using ultraviolet illumination on a
`Zeiss Universal microscope. Mitotic cells were easily seen.
`1,000 to 3,000 cells were scanned and those in mitosis were
`classified into the various mitotic phases.
`
`61%
`
`Illustration of the nuclear abnormalities seen in griseo-
`Fig. 2
`fulvin treated hyphae. Hyphal compartments with two, three
`or four nuclei were often seen. The Y shaped metaphase often
`encountered is shown.
`
`a cell was seen the two nuclei were always some distance apart.
`Control hyphal compartments with three or four nuclei were
`never seen. The presence of multinucleate compartments in
`griseofulvin treated fungi may explain the previously observed
`increase in their DNA content relative to controls‘.
`Griseofulvin also caused abnormal metaphase configurations,
`the most usual aberration being a Y shaped metaphasc plate
`(Fig. 2). Occasionally, a griseofulvin treated compartment con-
`tained two nuclei which were both in mitosis (Fig. 2). The
`radial growth rate of B. mnarum colonies was inversely related
`to the -logarithm of the griseofulvin concentration in the
`medium2 (Fig. 4). There was, however, a direct relationship
`between the mitotic index of the mycelium and the griseofulvin
`concentration (Fig. 4). The known mitotic inhibitors, vinblas-
`tine, sodium cacodylate and isopropyl-N-phenyl carbamate
`(IPC) cause similar elfects to griseofulvin on the colony radial
`growth rate and mitotic index of B. rmummz. Neither the colony
`radial growth rate nor the mitotic index of B. ranarmn was
`affected by colchicine or 1,4-dichlorobenzene (two other inhibi-
`tors of mitosis). Inhibitors of RNA, DNA and protein syn-
`thesis, cell permeability and respiration all caused a reduction
`in both the colony radial growth rate and the mitotic index of
`B. rrmm-um (K. G. and A. P. J. T., unpublished). The observed
`increase in the mitotic index caused by griseofulvin and other
`mitotic inhibitors was not,
`therefore, a general response to
`inhibition of growth.
`The number of nuclei in each phase of mitosis was calculated
`as a percentage of the total number of cells in the population.
`When these results are plotted as a histogram, a pattern emerges
`for the treated cells that reflects the point, or points,
`mitotic cycle where the inhibitor has most effect. We have called
`this histogram a plot of “Phase Accumulation Data”. The terms
`prophase, metaphase, anaphase and telophase are applied to
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`
`Fig. 3 A micrograph of a binucleate hyphal compartment
`produced by growth of Basidiobolus on a medium containing
`5 pg ml’1 griseofulvin. The micrograph is of material fluorescing
`after staining with acridine orange. Note the brightly fluorescent
`nucleoli, the nuclear membranes and the close proximity of the
`nuclei.
`
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`
`I ! J
`
`NATURE VOL. 244 AUGUST 3 1973
`
`from that of colchicine, should lead to its use as a valuable
`research antibiotic.
`
`K. GULL"‘
`A. P. J. TRINCI
`
`Department of Microbiology,
`Queen Elizabeth College,
`Campden Hill Road,
`London W8
`
`Received April 19, 1973.
`* Present address: The Biological Laboratory, University of Kent
`at Canterbury, Canterbury, Kent CT2 7NJ.
`1 Huber, F. M., and Gottlieb, D., Can. J. M1'crob1‘ol., 14, 111 (1968).
`2 Trinci, A. P. J., and Gull, K., J. gen. Microbiol., 60, 287 (1970).
`3 McIntosh, J. R., Hepler, P. K., and Van Wie, D. G., Narure, 224,
`659 (1969).
`4 Hepler, P. K., and Jackson, W. T., J. Cell. Sci., 5, 727 (1969).
`5 Malawista, S. E., J. Cell. B1'0l., 49, 848 (1971).
`‘‘ Mauro, F., and Madoc-Jones, H., Cancer Res., 30, 1397 (1970).
`7 Nordberg, B., Scand. J. Haema!ol., 7, 349 (1970).
`3 Margulis, L., Neviackas, J. A., and Bannerjee, S., J. Protozool.,
`16, 660 (1969).
`
`Antimitotic Action of Griseofulvin
`does not Involve Disruption of
`Microtubules
`THE antimitotic action of griseofulvin in plant and mam-
`malian cells has been attributed to a eolchicine—like disrup-
`tion of microtubules. Treatment of Vicia faba, Allimn root
`tips or HeLa cells with griseofulvin, for example, resulted
`in the accumulation of cells at metaphasem. The griseo-
`fulvin-inhibited cells contained abnormal arrays of chromo-
`somes, identical to those produced by treatment of dividing
`cells with
`colchicine. Using polarization microscopy,
`Malawista er al.‘ found that the addition of 10‘5 M griseo—
`fulvin to dividing Pectinaria oocytes reduced the size of the
`meiotic spindles, as measured by loss of birefringence. This
`action was considerably more rapid than with either pedo-
`phyllotoxin or vinblastine sulphate; moreover, on removal
`of the griseofulvin, recovery of the spindle also occurred
`much more rapidly*. Recent evidence indicates that several
`of the drugs which arrest metaphase interfere with micro-
`tubule function through different mechanisms. Colchicine
`and the vinca alkaloids, for example, bind to tubulin (micro-
`tubule protein) at different molecular sites“. Griseofulvin
`does not prevent the binding of colchicine to tubulin, nor
`does it affect the ability of the vinca alkaloids to stabilize
`the colchicine binding activity of tubulin5.
`This suggests
`that griseofulvin does not bind at either the colchicine or
`vinca alkaloid binding sites.
`It is now possible to polymerize microtubules in vitro, in
`crude supernatant fractions of brain containing tubulin, by
`removal of calcium ions by chelation with ethylene bis(oxy-
`ethylene-nitrite)
`tetraacetate (EGTA)7-3. We studied the
`effect of griseofulvin on this, and found that microtubule
`polymerization in vitro was not prevented.
`Examination
`of griseofulvin-inhibited HeLa cells by electron microscopy
`showed morphologically normal but disoriented micro-
`tubules.
`Ineubation of the crude 30,000g supernatant brain extract
`containing EGTA at 37° C resulted in the formation of
`250 A diameter microtubules.
`Addition of 5><l0'5 M
`colchicine, 2><10'5 M podophyllotoxin, and 2><l0‘5 M
`vinblastine sulphate or vincristine sulphate immediately
`before initiation of the polymerization reaction completely
`prevented microtubule formation.
`Polymerization in vitra.
`
`1 mn
`p m a
`Control
`
`1’
`
`’"
`
`a
`
`’ m"
`
`G
`
`p m a
`
`1 mu
`
`[2 m a
`
`1
`
`nm
`
`SC
`IPC
`Fig. 5 Phase accumulation data for control colonies and
`colonies growing on mitotic inhibitors, 12, prophase, m, meta-
`phase, a, anaphase,
`t,
`telophase, and mu, multinucleate. The
`pattern for sodium cacodylate shows the block of mitosis at
`metaphase. The patterns for griseofulvin (G) and isopropy1—
`n-phenylcarbamate (IPC) are similar but differ from that of
`sodium cacodylate (SC). Griseofulvin and IPC increase the
`number of metaphase nuclei but they also produce large
`numbers of multinucleate hyphal compartments.
`
`nuclear division in a classical manner. We have used a further
`term, multinucleate, to indicate a cell containing two or more
`nuclei each with an interphase appearance. The phase accumu-
`lation data, for all the mitosis inhibitors tested, fell into two
`groups, typified by sodium cacodylate and IPC (Fig. 5). Sodium
`cacodylate showed a colchicine-like effect
`in blocking large
`numbers of cells at metaphase and few multinucleate cells were
`seen. IPC, however, showed a significant peak at metaphase
`and very large numbers of multinucleate cells. Figure 5 shows
`how the phase accumulation data for griseofulvin closely re-
`semble those of IPC but are very different from those of
`sodium cacodylate.
`The nuclei of griseofulvin induced multinucleate compart-
`ments were always observed in close proximity (Fig. 3). In such
`cells the daughter nuclei appeared to move only a very short
`distance apart after metaphase and then reverted to the early
`interphase state by re-enclosure in the nuclear envelope. In this
`sort of situation griseofulvin may act by inhibiting the postu-
`lated sliding of microtubules“.
`IPC is known to produce disorganization of spindle micro-
`tubules in dividing plant cells‘. Our ultrastructural observations
`on Basidiabolus have shown that griseofulvin produces very
`similar effects (K. G. and A. P. J. T., unpublished). There is an
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2093 - 6/6