Document yLog8pm0DppOJ51dQXb0awY3

* rv v*#l m;* u- l..ivc .1 lii lii ill I'Milijpn itinn. Since (he side rlinms !nll\ r.vlniiliil .illi'i --|irt'inline on n ryl< liromi-c mu- 11. w lm Ii .if pH e.o must lx- poi-ifivt ly charg'd, it is lv fiat lln m(Ii- chain* repri-i iit pHvnnil'llic nv'le- FU-ci rut niicp'sci'pical oNcrvatinns on polysaccharide ,,t pn.;cin-)xilvi-,'<`ch;ui<lc coipplcxi s arr wry scanty. For i-.i.im t, it h,as Nth sh'wi that hyalmonic acid prepared .in human sviimi.al fluid represents .a linear molecule n-i!..'ii( lr.^lH'llinf:,". 1 li.wvvcr, iiranyl stained proloin; '.-.e ili.irirle ii-nple.xes piepared from bovine nnnl t.iriih.Ro have lieen lound to consist of a protein core fila ment with requlirly arranged side chains representing the .homlroitin sulfate molecules11. Although the structures nresent in M-DNA preparations show the same type of configuration, tlicy differ from the molecules obtained Irorn cartilage by the greater length of the side chains Six), and the size-distribution of the central filaments, n our material there is a continous distribution in length, whereas the tnacromolecules of nasal cartilage fall within two distinct siza classes, presumably representing mono men and dimers respectively. On illp t' i he -f .ircit \- of liic inn 11 r i;i I lu mi r j - o j, ., ,. liilux. elu-mic;il uh mifii ,itimt of tlu -.e nml* ul. pending. Ni t < rt hrlt***. tiie rr-gnlnr t fttrr iit r of it molecules in 1>N.\ r'.irarN of highly punfnd n it l.on tlrinl preparalions sucgi '.tc that proUitipol\ :u < l,;irn|i- complexes (pvntcogl-.tmis) may represent a Ui inline o.rn- )x>nent of mitochondria. This view is also supported by ffto finding that isolated milorhomlri/i of rat lo r ,,re capable of glycoprotein synthesis'* and tie nr. nt identification of the. glvci.pioteins as eonipomm- of mitochondrial :-l metura) pro. mix". Hxpi. riin< no .|. .yn. d to localise such moleculrs within structural r.nnipont nts of mitochondria arc now in progress. Zusainwenfassung. In hochgereiniplcn Praparntcn von M-DNS aus Mauslcber, die mittcls Phenol extrahiert und auf einem Protcinfilm gespreitet wurde, konnten proteo- glykanartige Makromolekiilc mit eincr Haufigkeit von 0,3% nachgewiesen werden. Diese bestchen aus einem Zentralfilament variabler Lange' (790-6220 A) und Sci- tenketten konstanter Liinge (1574 135 A), deren Zahl mit der Linge der Zentralfilamentc korreliert ist. P. Wellauer and R. Weber1* Division of Cell Biology, Dtparimtnl of Zoology, UnivertUy of Bern, Sahlittrasse 8, CH-3000 Btrn witterlarnt], 10 M*y 1971. Pig.t. Correlation between Um length of central RUmenls and ths aumber of side chains. Tbe data are baaed on measurement* of 26 trail dafined structures. The electron micrograph* were taken at a magnlflcattoa of 30,000 x. Tbe structures were enlarged to a final magnification of 633.000 x, traced on paper and the length detergained by tracing with a map ruler. ' J. H. Fesslzr end L. 1. Fesilek, Proc. oatn. Acsd. Scj., PSA 34, 141 (1966). u L. Rosehsssc, \V. Helimakx and A. K. Kleikschhtdt, J. biol. Chem. 243, 4123 (1970). ** P. tVELLAUaa, R. tt'EBEe and T. Wvuta, in preparation. " B. C. Westmorziaxd, \V. Szvialski and H. Ris, Science 163, 1343 (1969). u A. K. KtiixscHjitDT and R. K. Zahh, Z. Naturiersch. 14b, 770 (1939). .( u H. B. Bosmanx and S. S. Mastih, Science 164, 190 (1969). M H. B. Bosmak.x and B. A. Htuawoant, J. biol. Chem. 141, 363 (1970). v S. S. Marti* and H. B. Bosmakx, Exp. Cell Res. 66, 39 (1971). ** Tbe autbon are greatly indebted to Prof. E. Buboecke lot helpful tuggsationt and critical reading of tbe manuscript. This work was supported by a grant of tbe `Swisa K'ltfooal Science Foundatkm' (Project Nr. 3.248.69). The Metabolism of Biphenyl by Pttudomonas putido1 la a previous report cm the microbial decontamination of waste water*, containing aromatic hydrocarbon* used aa nuclear reactor coolant*, we have isolated, by enrich ment cuKnm, a strain of Pseitdomonets putUU able to , grow on biphenyl a* note carbon and energy lource*. In the present work research on tbe degradation of biphenyl by Pt. pntidm ia reported. MeteHmk tnd methods. Tbe cell* were grown end her- vetted and the cell-free extract was obtained aa previously described for Pt. drsmotyticum Proteins were determined by the method of Wsiavra and Christian*. The apeetrophotomctric determinations were carried out in a Zeiss P.M.Q. If. A model 137 Infracord spectrophotometer (Perkin-Elmer Ltd.) was need to obtain IR-abeorption spectra. Maas spectra were ran by a LKB 9000 spectro graph, TLC and CLC were performed as described in the experimental results. Biphenyl was supplied by Merck; 2-, 3- and 4-hydroxvbiphenyls by BDH, K and K Labo ratories and Eastman, respectively; 3,4-dihydroxybiphenyl by Eastman; benzoic acid by BDH. Each product wai further purified by crystallization. Remits. 14-16 h cultures of Pt. puiidM incubated with biphenyl showed positive test lor diols*. When the diol concentration in 20 I glass jars cultures was at a maximum, the culture fluid was collected and made alkaline (pH 8) with aqueous potassium carbonate. Inorganic phosphate precipitate, residual biphenyl and cells were removed by filtration and 3 extractions were performed with ethyl ether; the solvent was dried over anhydrous magnesium sulphate and evaporated in the cold under reduced pres- DSW 032773 STLCOPCB4016735 ttn k rrr nr. . ru snrr. The crude residue (20 mg) was dissolved in a lew ml were simultaneously induced to oxeli/v tin, ' ,mpoen,| t>( o. I ,V NnOH solution and re-c.\trnctcd with rthcr. The Catechol is oxidized by mWo-cle.-ii ag> mill in -nzvrr ethereal extract was evaporated to dryness and the crude diol wat refluxed with HCI in Aqueous methanol (methanol: \vntcr:conc. HCI ** 5:3:1) for 2 h. The solution was ex tracted sev eral times w ith petroleum ether (b.p. 30',-50,'C), I,system that is conslitutivrly present. Ct'Mchi.'-i'HH. Like other aromatic hydrocarbons phenyl undergoes oxygenation to a diol The diol i.ol.v, t from biphenyl cultures of Pa. putida is shown to h- t washed with 3% sodium bicarbonate, dried over anhy dihydro-2, 3 (lihydmxyliiphcnyl. In effect the ik-hed: i drous magnesium sulphate and evaporated, A portion of tiitn product of this diol was identified as 2-hydrox . in this oily residue was redissolved in water, the solution adjusted to pH 10 and treated with 2. 6-dichloroquinoncchloroimide and n-butanol. The organic layer showed phenyl A small amount was also found of a compound with the same TLC and GI.C features as 3-hi drove h|. phenyl. The further degradation of the diol leads to i;... f.mlu 635 nm. In the same conditions authentic 2-. 3- and production of benzoic acid through a series of unidente 4-hydroxybiphenyls showed 2,,,., (in n-butanol) 653, 673, tied compounds. 662 nm, respectively. 2-, 3- and 4-hydroxybiphenyls were Recently Lunt and Evans* found that a gtam-ne gttivr run on a thin silicm-gel layer, activated at 100C for '/ h. bacterium oxidized biphenyl to phenylpiruvic acid with Petroleum ether (b.p. 30*-30*C) and ethyl ether 1:1 were the intermediary formation of 2, 3-dihydroxybiphenyl and used as developing solvent. The spots, detected by spraying a-hydroxy-/J-phenvlinuconic semialdehyde. The meta with 0.3% potassium permanganate solution, showed Rf bolism of biphenyl by Ps. putida is different after the in 0.32, 0.33, 0.40, respectively. TLC of the phenolic mixture termediary production of 2, 3-diol as we isolated benzoic obtained by dehydration of the crude diol gave two spots: acid from the cultures and showed that the cells grown ox the larger one showing Rf 0.52, the other very smell with biphenyf are simultaneously induced to oxidize brnznn; Rf 0.33. Gas-chromatography of 2-, 3- and 4-hydroxybi acid in Warburg experiments, not phenylpiruvic acid. phenyls was performed by a Carlo Erbe GT mod. 200 Gas- Only the cells grown on phenylpiruvic acid arc simultane chromatogTaph. with a flame ionization detector. Column: ously induced tn oxidize this compound. stainless steel, 2 m x 2mm I.D.. packed with 10% SE on Research is in progress in order to clarity the metabolic Chromosorb W (60-80 mesh). Temperatures: column 210oC, pathway from the diol to benzoic acid. detector 230C, Injector 2J0C. Carrier gas nitrogen at 1 atm (40ml/min). Hydrogen at 0.8 atm and air at 1.4atm. In Riassttnio. La degradazione del hi fertile da parte d these conditions the peaks of 2-, 3- and 4-hydroxybiphe Ps. putida inizia con la formazione di un diolo cui si attn- nyls showed retention times of 3.75, 4.40, 4.45 min, res buisce la struttura di 2,3-diidro-2, J-cliossihifi ndr iq pectively. Gas-chromatography of the residue from the quanto, per disidratazione a caldo con acido t ioridricn. -4 acidic treatment of biphenyl diol gave two peaks at re trasfotma in 2-ossibi(eni!e e tracce di 3-ossibdrnd- I) v tention times 3.73 and 4.40 min. relative amounts diolo viene ulteriormeote degradato ad acido benzon < 93:5. The mass spectrum of the first compound (m/< - 170) Le cellule di Ps. putida cresciute su bifenile sonn simul- was wholly superimposable upon that of an authentic taneamente indotte ad ossidare al Warburg il bifenile e sample of 2-hydroxybiphenyl. The spectrum of the second 1'acido benzoico, non il 3,4-diossibifenile e l'acido fenil- component of the mixture (mjt -- 170) could not be com piruvico. Le stesse cellule contengono una metapinx.ite- pared with the spectra of 3- and 4-hydroxybiphenyls casi costitutiva. owing to the poor amount relative to the bleeding of the column. D. Catelanl C. Sorlini and V'. TreccanI In other experiments the mother liquors from the ex traction were acidified to pH 3 and re-extracted with Istituto di Mierobiologia A grant e Tecnica, ethyl ether. The crude oily residue (70 mg) was heated1' Universili di Milano, lTo Ctloria 2, with a few ml of petroleum ether (b.p. 30*-30',C). the li 20133 Milano (Italy), 30 April 1971. quid phase decanted and the solvent evaporated. White crystals were obtained after repeated crystallization from x-pentane (m.p. 120'C, undepressed by admixture with authentic benzoic Acid). Elemental analysis was as follows: 1 This work wat tupported by a grant of C.N.R. (Comigliu National* for CrH,Ot calc. 68.80% C; 4.92% H; found 69.14% C; 4.93% H. IR, UV and mass spectra were identical with those of benzoic acid. In Warburg experiments, cells of Ps. putida grown on biphenyl were simultaneously induced to oxidize biphenyl and benzoic acid, not 3,4-dihydroxybiphenyl and phenylpiruvk acid. Only the cells grown on phenylpiruvic acid delie RIcereHe), for the International Biotoiueal Program- D. Cartt-AM, G. Mosszlnass, J. Niesiials. C. Sorlini and V, TazecAXi, Experientia 24, 922 (1970). * V. TazcCAKi. K. Galli, D. Catelasi and C. Sorlini, 7.. all* Mi* krobiol., 63 (1968). * 0. Warburg and \V. Christian. Biochrm. Z. J to. 344 (194 U 1 E. Borland and G. II. Wiltshire, Biochrm. J. 5J. 424 (1933). D. Lvm and W. C. Evans, Biochem. J. //*, 54P (1970). Zum phototaktlschen Laufverbaltan junger Honlgblenen (Apis meltifica L.) Bevor Stockbienen zum Sammcldienst libergehen. machcn ie mehrerc Oricnticrungsfliige. Sie beginnen damit etva am 5. Lcbenstag (RGsch*. Sakaoaaii*). Eigcne Bcobnchtungen tind Kxperimcnte zeigen, dass junge Stock bienen vor ihrem 1. Orienticningsflug ebenso wie ausfliegcmle Sammelbicnen (jACoas-jKRSEN*) pnsitiv phototak- tixch gestimmt sind. I'hototaktische Laufversuchr sollen zeigen, ob und in- wivucit sich in don ersten Lelwnstagen adulter Bicnen die Uenittion gegenlilK-r {.ringon LiLhiintettaitaivu -1 :t*t -- " nirnert dcs 1.-6. l.ebenstagcs wurtlen nut Tngv in t-nf t vcrdunkelten Raum (bei 2b "C) mit einer Versuchonord- nung getestet, wie sie in Kigur 1 dnrcestellt ist Itnih ScttRlCKER *). Eine positive Rcaktion war ein tinimbr- brochener Lauf durch das beleuvhtete Ruhr AI-< ' 1 r suchstierc dienten um 0 1`tir 2 h im Mrutschrnnk yeschlitpftc Bicnen, die zu dicsor Zeit bei schr schw-whi ni Rutlicht gekkfigt unil not cint-tn I'ollcn ITittt'rt-'i-.'ci - DSW 032774 STLCOPCB4016736