Screening Microbes Isolated From Melalap, Crocker Range For Inhibitors Against Both Prokaryotic and Eukaryotic Signal Transduction and Isocitrate Lyase In Mycobacterium

Jouwer or Tnoprcer- Bror.ocy Aro Coussnvrrror, 2 (1) : 27-50, 2006

Research article

Screening microbes isolated

from Melalap, Crocker

Range

for inhibitors

against both

prokaryotic

and

eukaryotic

signal

transduction

and

isocitrate

lyase in Mycobacterium

PU.dH S€ok Hw4 HEW Chaw Sen, F'OO SekEin, ONG Si Mon, HO Wei Loon, LEE Ping Chin, LIM Siok Har and HO Coy Choke*

School

of

Science and Technologt, Universiti Mal ay sia S ab ah, Locked Bag 2073,

88999 Kotq Kinabalu, Sabah, Malaysi.a

n

ABSTRACT. In this study, 65 soil samples

from underneath identified plants were collected upsfeam along the Melalap river.

One hundred and thirty-six actinomycetes and ten microflrngi were isolated using selective isolation methods. These pure isolates were cultured aerobically for secondary metabolite production. They were screened for inhibitors against three yeast-based molecular targeted screenings: protein phosphatase 1 (PPl), glycogen syrthase kinas€ 3p (GSK-39), Ras/

Raf-l protein-protein interaction, aad two Mycob acterium-based screening systems:

isocitrate lyase

(ICL) of

the glyoxylate pathway and PhoP-PhoR two component signal transduction system. Three extracts (H11329, Hi i337 andHl1402) were toxic to yeast in Ras/Raf-l screening, nine extracts were toxic to yeast in PPl screening (Hl1293,

H11298, Hl1300, H11301, Ht 1302, H11304,

Hll3l7,

H11339 and H11402). One actinomycete strain Hl1299 showed weak inlfbition to PP I . Two extacts (H 1 1 329 and

Key words: Actinomycetes, microfungi, two component signal hansduction, protein phosphatiase

l,glycogen synthase kinase-3p,isocihate lyase

.:

hocoychoke@yahoo.com

II1 13 64) showed weak inhibitory activify and

tlree extracts (Hl1339, H11337,

Hll402)

showed

toxicity in

the

GSK-39

yeast screening. Five extracts (H11310, H11317, H11337, H11346 and H11383) showed toxic effect in the ICL screening system, and one

extract (Hl1392) possibly showed weak inhibition to the PhoP-PhoR two component system. It is interesting that Hl ¹³⁸³ has th€

same inhibition characteristic as H7763, a presumptive ICL inhibitor with a wide partial inhibition zone on acetate plate (Dain, 2003).

INIRODUCIION

Actinomycetes are common Gram-positive bacteria in soil, composts, river mud and lake bottom. Rich soil tends to have a high yield

of

actinomycetes. They are characterizedby their high G+C content and cell wall compositions.

Actinomycetes together with fungi are avid producers of secondary metabolites with classic examples being streptomycin and penicillin. As chemical novelty associated

with natural product is high, metabolites produced by bacteria and fungi provide great stmctual diversity (Hawey, 2000).

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litters were collected aseptically into sterilized bottles with th€ aid

of

sterilized tongue depressors.

Soil pH determination

Ten grams of air-dried soil sample was rnixed with 25 mL of distilled water. pH reading was

taken using a calibrated WTW

inolab

pH meter.

Isolation of actinomycetes

Actinomycetes were isolated by plating out diluted soil suspension onto humic acid B vitamins agar (I{V), arginine vitamin agar (AV), starch casein nitrate agar (SCN) or diagnostic sensitivity test agar (DSTA). Cycloheximide was added

into the

media

to a final

concentration of 50 mg/L to prevent fungal contamination. In some cases, pre-heatment of soil samples with chloramine-l phenol and

D

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0- **l_,kt Figure 1. Topography map of the study site. Contour lines are in feet.

+

N

l$.*ll.d r....il! (1! &Lr[a)

trLl:r.itt"25. Murl$1t52 urL7.,t r.!0, Mu.tlalrar,

heat treatment at ISOC for two hours was done. Distinct ^colonies were streaked onto oatmeal agar (OA) for observation.

Selective

isolation oI

Strcptom!cetes (Nonomura _and Hayakawa, 1988)

Soil ^(0.05 g) was suspended in 9.95 mL of

distilled water. The soil suspension was mixed by using a vortex mixer and ten times diluted thrice. One hundred gL of the final dilution was plated onto HV agar (humic _{acid 0.5} g/L, Na,HPQ 0.5 g/L, KCI 1.7 g/L, MgSOo.THp 0.05 g/L, FeSQ.THrO 0.01 g[-,CaCOr0.02 gl

L, thiamine-HCl, ribofl avin, niacin, pyridoxin-

HCl, inositol,

Ca-pantothenate, p-

aminobenzoic acid each at 0.5 mg/L, biotin 0.25 mg/L, cycloheximide 50 mg/L and agar 18 g/L) and incubated at 28"C for 1-2 weeks. Distinct colonies were plated on oatmeal agar (OA)

(Quaker oatmeal 20 g/I- andagar 12 glL).

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AbicedlbdfuFni4

5 wL36 ,nrue ^3Fe!Mhg) 37 MLL3? o.m,.',ffi

^(Bisdg)

^ ^a s MII3I rbur@tcdlhbbbn)

A

15Ml'45Mqd]A.oD\,'M

e[sbupi6el.f'

aaLw,dfuliado

5, e52

GlhD

35MUJ'^fufupafub

fiow'InaLEEP

^c

Cn,I-lM SdfiRmfiOCdG{E

4h|furF*!uornE ,{ayfudwd6edd

awyfudqWo'r!i3l'l

b*.nlul6'dofnu.,|l5o

6t MLtj3 storuNrcinuo

6 jt,nt-fs Uiwniodada

^sp

SqBNo MgoB

^FRoM

^Idl'jMP

^ER

I^BrcB

tlDle 2. Lid ol mrinomyc.B

isolr&nnu sil tupler orM.lilrp

su

rb{. oNG s Motr, HOWfl

ljd.

^LEE

PN CN.

LIM

SId

SffiCMlxo8 FTEMEAre M b|Drci9

B4F

.rdrrrh

^Nq

.l3ltydlryNo

rilh Fl

LthrFrbv

^No

PUAH smx

sva

HEw

c,

wSs.FOOS4HN.ONGsrMoN, no

wErIrN,

LEEPM Cm, ^LIM

sox

Trbr.I M'mtune ilobd

^from M. ,l,p ioil

lmple

.oroud..redD'whb€smM'udn.mcorountdrid2cq'loserNipf.nfrl

2002)

'Ibbb,r.

R.:ultsfork R. reicD*in

HrLlGHm2r 0

⁰

Hll3?9 3mi(C)

^&m(C)

srlroHm36 0

⁰

0

^50om(D

Hx.a& hs(nv) ^hm(\r')

Fr@drLd.fu(chqinl,intu.}

HlBrlqil- lomo ^lrm(c)

Inl3trzqrl- l4rm(c) ^lsm(c)

I|1l3t3q'! l7m(O lh(c)

HlBr4q[ tu(c) tu(q

0

^No itr1i6idon

@ @bd

^prpd

di$

P

^P.nid

^i.nibirid

^{shovins rftrc}

_sdb ^ofy*r

^efthin

^{iihjbirih ed.}

W w..l

inbibirid shdjns

-;r-'r r€d

raEtiodvirhin inbibirio

zd.

T!br.a,

Fs,irdidac..dvi9olLzell3lftd ^vj$ n@di.ddtu.olHI

I337.

MP ]ldit _*p{omr.diu

PUAI{

Sd Iira

HEwCll^wSb. ^FOO

se sa

ONG$

Md, fiOwd r!d. t-EEh€

Cm. ^LIM

srd HrN

Ho cry

rion

Trri.6, n6ur6rq

PPr

srqiig

---!Ll(Ei!4@t!L ---L .@-!!64!l

0

^{No iobibidd}

^{,@ mMd}

^papd

^dic

P Pdidilnibirid oinglt6r.gEerhofysrsdmifibirimb..

40

lll or.idd

^is rppli.d ^on

th

^dis

s.rMcMrc@sIM I'/txswm ImFoB

rrbre

?..Rdr!

fdGSK-3F

smin&

H|2n-E

^t1?8

IIt14@

0

0

^No

^ioiibidd

^zotu adtnd pap{ d&.

w w.,r.idibidorrh*j4sihifl,lycarEdu.timeiininidhibiridbft

40 pL

or.xtu

ir aDDlGd on

ah di*

:hbL7b.

R6ult

Io.E11t9.

trn3?,8rr339,81364 Err{02Ri.h1mFLor6d.rdd.

P

^Pdiar

i,nibi.rd shwi,s lid. si!*n

w

^Wa*

^{iobftiio $dwir!} -nhi r.d

Edrcti@ wirlin

iohibiiolao.

HO

WEInr,

L€EPnoCm, LIM SM

B,um

HOCd

Cilm Ahi.

&

R.$lb lorMy..6€cedm

Ic],

maning.

n3om,,@rd.rrd r@dt!

^ECrs5

Hllt46

0 0

P PMidiinibirid

^sbowbs

liri. g6{d ofntprocrd@

^wirhb

titibidmzG

-4o

IL ord@t

i5:ppli.d

o €b

di&

S(]r:EmordxroEhdl,Irlj4nlrlBlBmtr

⁴⁵

rrhl.e.R6ullrro.itrordk*ldPtoP-Ptdrt'o.modMts'sLns.ruhs.

g,,'0o,

Mlt

o

hdqi6 il. stuir ne

^{ts s}

0 Noi ibidd

rod.

3fud

paFr dilc

20 d-

orexhr

i6 ippri.d

d

^ei.h

dit

fttte10. cmdr'rddd.ldd.rcjofsl'dtrIItl3Din15,t[l'$ti4mrMudrnnof

t0

ll

0 .oiDbibirimzuetouldPiFrdis

PUAH

S4

Htrr, HEw CH^v

s*,

FOO SFK tll|, ONC Sr MoN.

prpdr

di$a pip.rt.d * crtrds ed.

Fcorded

afh

lwo days. Extucb sho*ing

inhibition only

oo

low

concentation

RASUUTS

sixtyfivesilsmpt4veE.ollsbddunng

rhe

dp.dirioi

13 smpl6

{eE@lled€dnd sitA.'sn'pIdnmsileB.14smd*lM

€ilwl)

^Th.

^so

^{plr in sic Antrged}

^iron

sib D:4.s15.47. sire E: a.m 6.93 ('&ble 1).

Th. sMy

3ib

lhdw.n _rypiclr

chsderinics ,b?o

^sp., ,|{arl,4r sp.. with only rew

Fi.!r

colleEd

sil.

Ninety-louofthe

islarc wF

sunj.c!€n b ^phmol

pFeMo!

l

lisoldd w.f fion

DsrA. rdur

roraB

^yed picked

f,mAv ii

whi.h loir

.dple, ldryenr h.d

pE beded wi'h

chlomin+r

^(rable 2)

dd

rn niciortrigi w*e

^isotar?d frcm sone

Ii

lhe

erceiinc, dE? errads

^(H11329,

Hr B3? mdrl1,()2)

w.ardi. by*rii tu

Rdll.eonu.wheHllr'washoMm'

hkridiic codd not b. Gp.abd {hen

rer.d

in a Enes olconmftrions ^(Table 4) Hr1337 k.ond

t9ffi

^s.ne,

tu.zin

iihibi'ion or G saladosi&se

sy

heds (Table

5)

Nine

dlds

(Tabla 6)

wd

^roxic

b

^y.ar in PPI

ffiins(HnDr,Hl1293,H1l30o,Hll30l, strl02, Hll304, Htr1r7. Err339.,rd

H11402). olre &tinonyere shin. Hl r2ee, shoved

qc*

inhibition to PP1.

In

tbe

p(rifrii,ry

csK-3P r.r@ing r@r

4@!

(El r329, Htt339,Hll364,Hl

1402)*,*ly

(Hl1337) _show€d bxicity

byeai

_crrble?a).

inhibily.*tdily,ll7.cvh4dHl]339'

Hr 133? md H1r402

iihibir.dy*tsodh {

borh 2s"c ^and

l7'c

(rablc',n).

Elrtu (HX3lqEn]]7,Hl

r33?,Hr 1346,

rdd tsrr133) showed

roxi eff.dl i

^M

rr?3m!i' lc,- $enins sy$d (IrbL

3).

H I 1333

,l$

sho*.d a vid. pdial innibidoo

(Hllrg2)

_shos.n

bxniq b rt eesmri!

cobr.9)

th.

^gswrh

idihrid

orHt r39zw6

l.sirhthmsgd.simm.diunecomp

ed ro low masncim medium (Tebler t0,

tl)

Fotri.irsds shovd,crivit il noE

rhan orc s.GDing

sy$4

^Hr r3r?

(oiic

in PPI md toxic in IcL). Hl ^l32e (bxi. in

R,iRdr dd

^po$'brE inhibiro in

cs(

38), Hr 1337

&nc

in ReaRaf-1. CsK-r9 edIcI-), Hl1339 rbricin ^csK,3tl,

di. i

^ppD, Hl l4o2

(bnc

inR eRrr l. PP13ndcsK,3p),

soil pH Eadings uken indimrd$acoil rmn

sitcwsmmeidic*jthvalresrugh3

3 63-{.6X Hr 1337,H11339. ardHr 136L *hich

colled.d

fion sir

c. ^As icljnomyces

e

(Holt

r 4l,

1994), the enumeration or bioacriviry

prcddi*

adinomycees

rDn

acidic eir is orinrre*.

ft.r

^i3,

io*cvc. se

posibility rb

^rbe isol,riotr _modidhav.

resticr.d cowrh ol

sne

etioomymG

it

ddic

conditioi

s 64

^aE

Jrepr,n)..r

bbrd

tug.

ofpH in higrr

dsric

load

mdia

(r$tu,r,r,

2m5),

Mos@riiom'€b wft

isorded

ron

so smpres.o

erd

6'm

sib

B, sn of $en showed $tiviry h

eEqing.

Sir oui or 14 isolares

sle.bd rbn

DSTA

ftdim

^sbdeed

bxic .ffed b![ds

^PPr

eennu,rs

^{y hieh a mmeff.n @}

doni*

isll@d

ftld Irv dd

oths nedia. Tnous!

biolosisly divc mhboliB,

rh.

!@i6c

lnoh

Soi!

reF

monly oned€d undd bis ,Jrar

$. nimbial comldt

conposirjoi

aMyd

^.r

,r.,

^2001).

ftc

r.pon of high.r

n1d*r6o,aodr;,bd

sils aYu.20Xl

sd

the

.$oci.rion! of ditfer.n.

micrcbirr

confunni.s rirh dirfereit

gra$laod

olmunity

iyper (Mccu _{.y &} Btrrrr., 2004)

eddPl6oflheeflebo'di{ldhAbibb

on

ni.sbi,r

rbud,ncE

fd

diveEity.

''e3,a!r

^md

^{bncny o'}

^rhe

^yeat-bs.n

R.s,/R!f-l pEaii-pto€in inGractioo

ffi

^d.

rxe nm.sp..ific

srioi qs

_!\en

_coolllmd

by

mn fdrrion of d. o,caraaordas

as Escd by nsi'g HPLC fmdons (d!a nor rhown). In

yed asK rll

sceninc, ^Hr 1329

rdH

t 136{onlyBux€dinp.sitive inhibnion high

conc.fu!.ion (r00ul). Fu

her

asK

39

nr,jbibn nieit

be uctur

ii

r

cw

rhenpeuric rppncanotr

rre lHo$

sysrm dtuodB, lypc 2 dhbcB.

.der sM-.en

biology dd

ncddari€

n€dicine

(Meijd,r ,r,20o{i stooF

& $bodFtr, 2003). RAs

MA?K inhibiroN

my

aho nnd rppriqrions

in

c,ffilnedpy

^(Hoben 3r,r.,2noti seb.n- L€pold ^&

Hefts,

2004) G.ldrndycin lnd

6di.i.ol4 tu

*amprs

ofindielR

!,Rrr innibirG of

ni.obirt

ongini b.rh

deae R

^{lwel by bbcriig} de Raf-Lsp9o comptex {suncab 4 dr, 1997; sogr d ar, r993),

A nor

specific

Pilinnibiioi

BAY 13 9006,

n cufttrdy

uidssoins ctinical niat for rbe

deyer@enr ofsn

sri-cserdna G,oa,!

oa, 2001).

FKJ06

d cyclGpoin

c

^rwo

imuoosnppamt dngE

6g.td

ar PP2B, a

Pro'eir

phosphabse

(cobcn,

200r).

N.@logical disordd, nEhboli.

disd*dd .ricq

hav. ^been a$ockted wirh pioGir pbosph,bas (Mcouksy 4u r,2002t

ioe

porenrhl druss rarsering

protein ruh€rculosis is srirl

rbreddiis lil

^dGpire

rhe

rv.ilability

or

vdous

dnss.

cxftdr

m mtim,r rB drus G

h@ry

etr&riE

sgains erively rplicding cans, bur dry

*

res poEnt ssaibi

cdk

in ddiooa'r pbae,

when riey

c

^capsble ofp.ais.itrs iftide lne huron lma (McKinney 2000). rhererdc.

inhibnos

hg.ti's ldcil

peBieor T3 is or pobnrial u$.

nr,r,.r?ri!d lcl,

and PhoP PhoR

ire

such

tacro bn,. i(r.4!/o'rr iit .tidr

(McKmcy

a

ar, 20m! FoMn ^g!

Th.

lefch

for bioadive m.r$oinos

ha pan

rwo decads.

Tb.

'pplic

^ioos

^ot

recorbioror

DNA

conshdioi

of

$enins systu a

in de

RasRdl ffi

^hd GsK-30 mrde Fssibl.

wirh

'ewq

phtrneoroeical *iowtedsq d)e

.xprnsion ro other

nicmbi,r

sourc*

hd non

^tibiotic typ.

mbbolirs,

hd embt.d

sinplified

^d

cor

enicient nc.hods

roi

novel conpounds di$overy

(obuA

1992).

Mtr

rhm hdf ol rbe knorD

sribions @

or ni.robi.l odgn,

PUAHS&X H{A,

HEwcs{s,Foo

HOwE rroN, LEE

Pnccm,

LIM s'o

dl'llmbi.in, dosdbi.ii.

dd

ftibnltin @

inpoi$r diobd

^{scehr (Dcmin, teee).}

an

invsdigarion by waog

4

4r, ⁽¹⁹⁹⁹⁾

rev.rled

_!he

ieol!rioi _of

₃₆

cei.a

^or

vith s|rcplonyc.t,

Naca.dia, and

st pbsro4tsinn 4 ^ij\.

mon

$uida ^ink

cives

r

€pscnration

oflhcaclinonydediv.6ilyiDtheftpi..l

ni!

toesL Cdedr

lno{d

didobc apei.s

m in!

^{a dny}

^tucrid

^of rh. wbor.

'.imbior dirnity.

They arc valuabl..e.ources in bitrchnology develoP@! dE

@sffrid

or nicrobi.l ^gerc !.ols ^is

6*ndrl

ha b

ond

ovenmbd @un.,ur, ^{2000). saba\ bei.e}

m

df

ft

^{wodd s} biodjwniry hobpo6, offc6

Range.

p.elimio

y

€sulb

indicate

rh.

isot.rion or

I nmb*

of

nFin, *i& w.d.

indud. two

!r,i6

otadinomycd* (Hl l3?9 8ndH11364) inhibirinc rh. csR.3b

seni.c

sydem. and one

shin

^{(Hl t2ee)} rbd innibirs

@tiMye&

^{(H r 1333)} b.hiv4like H7763,.

pasunpriv.ii$bndorIcL$r6ing rysh

(Drim, 2003). Funhemore, rio'her n'issddyemphaiz:sdEu'rlzdimofbgn

miftbiar divssi'y

offtdmmyEB

ad tu@

in the learch for

^Yahable secondary nctboli@s

foi 6c

^poonlial

rE

nenr or

diserses

d Bberculcis. ^-rbe exr.osive

cm.rer kng. Prk

^(r39.9r9

h(6s)

in sabaL wirh vaned bposnphy rnd

wgdtion

ryp.s,

n

a unique

biorosicd

resource

qdrinq

inv.niadiotrs

ud cor$ddion.

we wotrld

lle ro.xpes

dd

ipptui,rid

rd

con$niton

lor rh. invidion to parucipie

in tbs qp€ditjdi.

sd

ro M. Jonnny Gi.il tor

hi

^{h.lp wiln}

jdcitiicdioi

_of

_{de phG.}

Dolois

subitr

^(our _sdded ror

$et

field

d!i.rd... rni! p6j.d

b tDnd.dby

e

IRPA

gfuatrmMalrysbnMjnistyols.jdcq lbcruorosy

d Envircl]lMr (01{210i033- EA0036)

dd

Utrive$ni Malaysia Srbih

Fn'drb

.nn.+@hcmtB-0901'PRru0lI

^dn

^{, P.D. & MJR,}

^{Sb*, (m).}

^{It?c r}

e(bwF4 ^Qrctkb. tund

ol

cdt

Bulr,^, T, .19c, wgd & M. e.odre[o*. ^(2000).

M.hbbb''dMfueblBbhDR.iq}'

N.*

& C.C, Eo. @04).

Sdrcii8

ror

hhiboEdotqodcM^P(liec

(M(Kr), Prcrh ^phNphd $

^(Msc5,

clcr)

@d

Mysbedd iNnuD

1yc.

(rcl) rdn

dhonye@s ud

fihMsus fuogind{dnoec@bfuq.,.!j47.

& J.

N!

^(ds )

cN.r4.4r!. sd?'ar.

tutoen rtudat

ol

eathxtrr, 26' coi.q

c (401),

ftorii ijis

^{r[. n.jor d^s}

64dld[.sdly.trsl..Uy?'d!E D!oi!. a.L oret Phlnlq'rdry eiiw

ANlEdMENbplwqdBbl.ch4alag'5z

DdD! s

(4Gl

sEniigformiqobdL iinjblon

Fo s.

qA., s.

wfuE

^{& r.} sDrd. (1004).

hsgip'jonal!4!aioohmFbaddim

cxbd.tr,

E.^,

(r00t,

Th. phioropt _rvo.

conFild 4uhbry

^{sF cn PhoP P|oa.}

b i d

af 3 @ ed ot @, ^t

I

^{I rA 5}

^r v2.

H,R4^.e000r sbt3i.! rordk6Y.iqdqs rremNoBLyumxdtrdIfula!d!.6

rr.obr., ri

^w,R.

_c.00). rrder4d4i4'

ra@t$.

b. Gds.) d.ebr

G.n4bs aJ

,t@ed44.

^sM

PE

*w4hi4bqD c

xi's'w'Kbhl4J'x'('o!',,.Elslnq s uoilo!

cLr. 0 rr3)_

tddrifi*riod

of

{e4itc,Fta run d

o1

t*ibiard,

NlY ri!!!.

(r00sl. _PH en

d,

^otr r0

c.c.

Ho. Ooott

AdnonFde iekrd

^ti.(.ds ^).

) scic ilir Joun4

^fhro'gh

NdgdE@ryluadse4l4cdPn5ll

r &

a M.c[*!y,a'A.Trsio& k ^$k0r

o001t

lrDak. orf4

^{APerrnbi da ndti}

ot t\6e6i. stu _si6 rawd at dtu.t

shre

& s.r

wnLn' oeq), 3?4r

^s

@utoltulq

hdb?

Rektida* pr d.lw li!tu&wiMm.BdriftE.

rdtqnd, B4 Bde,

^R. c@dr &

L

kPp.@0q),

cdni Meritu@d'ic tuur otcqdr@n6n,3i-6.

Phift:hids4.vtr$ltgjddYdlbiry'

sor sckde sd.bo

7f)nqia ror d,

PUAHSFXHv,HEwcu*s,Foo

HOws l@N,

LEEPmcm,

LIM Snx

Hdm

Hocoy

Olm

M.KrDq,

J.D. r(.

Eo!!i a r!tup,

^E.J.

Mnd.Elu,A'M6L'Be!qa

R- J3cob,.

J.

^&

^D.q nK.n,

_@00),

nm!ia36ddfu.quft56c8lyqylfu

Mlij4

L., M

nrjdd

& P

ce4'd.

^(!00!).

Phunl.ologicdiiblblofofglyco3en

*fuEnale1'TI..,d'kPbfu4l4i41

Pa.dr.@oo. ui&aPtd

tsnms

d so sd.ru srkt!.t

^ntxo ^towrd, NrpFr

P!iD1. (1002).

Mnbuiun cdou!

Nmonun, E, & M,

thrrlor& _o'3&.

N.w

rliimyeeiP'.a3,93hokmi.Y' 844,a

sdQaw@.

s

^(ds

)br"3ral

o|ib, w,N,l1t6rl E lnnidorNaa'pd

oorn,

^{s. (ree2). rrcld5}

^h

^rb! &nch ior

bberi& aibbbr n.ubrftr /ol@r d

s.bon-rdp0rd, J.s. &

r. Ecrcn.

(20M),

a4ed4eniq{fuqdpddbsl

wR. Ji

^&

srccrdriDi, J.c.

^(1000).

h.M.t W ^rtr

^d

^r'

^{!@ 1r b}

^ti.

^d

^dn

^Nd

u.

sdel s,, T'

xoaq

^H.

N.lml s

^r1n4q

lirlcDqT.MhL

^a

M,ILn

Ot9r).

3i@ri4

^p.'hrry.

r,lriar

a/

r,rrr3'.or s@{bi.c,,

E,c

v!sd!

!,

stuoq

e E,^.

cdhm _oeo, Mddde Mt

^df

d.

sd@dhdPrinunw:ld.nli6c'n66

tueEgdsB.kwt6tutut@,

srrsft,

^{LF, a,M.}

strl*b

J.x.

tu

oeer). rh!

hlpeo-bhdh8

a ibbti

dinpdg tudo! d{Ftug

a@prqB

F{n\u. tund

^{otRid4nd chznnry.}

sbootsr. ^{3. D.} & t-

wdsdl _cmr.

M6br

€.M. !r. ^(reee). hvsris,hi

^or

niirmbof sie.po€.rq@btz?drbr

ri

^{z,! G}

f, s.n.

^&

r, w Mtu, (rm),

edcbdd5*6'd?9fu!dw@idl