Biologically Active Substances of Protozoa

121 Pages·2003·2.373 MB·English

by  Natalia N. Sukhareva-Buell (auth.)

BIOLOGICALLY ACTIVE SUBSTANCES OF PROTOZOA
BIOLOGICALLY ACTIVE SUBSTANCES
OFPROTOZOA
by
NATALIA N. SUKHAREVA-BUELL
Academy ofTechnological Sciences of the Russian Federation.
Moscow, Russia
New York Academy of Sciences,
New York, U.S.A .
....
"
SPRINGER-SCIENCE+BUSINESS MEDIA, B. V.
A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 978-94-010-3787-7 ISBN 978-94-007-1088-7 (eBook)
DOI 10.1007/978-94-007-1088-7
Printed on acid-free paper
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© 2003 Springer Science+Business Media Dordrecht
Originall published by Kluwer Academic Publishers in 2003
Softcover reprint of the hardcover 1s t edition 2003
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TABLE OF CONTENTS
Preface vi
Acknowledgments ix
Introduction xi
1.Protozoa as producers of biologically active substances 1
1.1 Trypanosoma cruzi as the sourceof medications;
selection ofother species amongflagellates 1
1.2 Toxins and detoxification substances 4
1.3 Biologically activesubstances of soilProtozoa 6
2. Cultivation of flagellates 11
2.1 Mediafor cultivation offlagellates. 11
2.2 Physiological role ofmain mediacomponents in the
cultures offlagellates 12
2.3 Conditions for flagellates growth and stimulated
biosynthesis of lipids 15
2.3.1 Inoculum quality andquantity 18
2.3.2 pH and osmolarity regulation 18
2.3.3 Influence of temperature 21
2.3.4 Passiveand active aeration 21
3.Lipids offlagellates 24
3.1 Phospholipids andsterols 24
3.2 Fattyacids and conditions for stimulated biosynthesis 28
3.3 Biosynthesis oflipids byflagellates 31
4. Glycosylated lipids of flagellates 36
4.1 Glycosyl-phosphatidylinositol (GPI)andrelated GIPL,
LPG andLPPG 37
4.2 Biological functions of GPIandrelated glycophospholipids 41
vi
s.Surface membrane glycoproteins offlagellates 46
5.1 Variant surface glycoproteins (VSGs) andtheir genes
rearrangement 48
5.2 Sialic acids and trans-sialidases 51
5.3 Membrane mucins and mucin-like glycoproteins 53
6. Cytokines, eicosanoids and nitric oxide as effector molecules
against parasitic flagellates 55
6.1 Cytokines 55
6.2 Eicosanoids 59
6.3 Nitric oxide 64
7. Biologically active substancesofselectedflagellates 66
7.1 Total lipid fraction:correlation betweenits composition
and biological activity 66
7.2 Astasilid,its composition and biological activity 69
7.3 Membrane glycophospholipid (GPL) andit's biological
activity 74
7.4 Reserve polysaccharide fromAstasia Zonga andit's
biological activity 78
Conclusion 81
References 87
Index 107
PREFACE
The search for new producers of biologically active substances (BAS) againsthuman
and animal diseases continues to be an important task in biology and medicine.
Experimental work must be carried out well in advance of need because it takes an
average of ten years to develop a new medication, as well as additional time to put it
onthe market.
Study of the Protozoa forms a special branch of biology - protozoology. The
traditional fields of protozoology are taxonomy, phylogeny, morphology, cytology,
evolution, ecology and host parasite-interactions. The Protozoa is the only taxon
among the microscopic organisms, whichhas notbeen persistentlystudied asasource
of BAS. This book then is the result of the research on the project: "Biologically
active substances of the Mastigophora (Flagellates)". The research was carried out at
the Laboratory of Antibiotics, Department of Microbiology, Biological Faculty of
Moscow State University. Articles of other authors on the matter have been
consideredastheimportant partofthis reference book.
The goal of the reference book is toelucidate scientific approaches, which lead to
obtaining biologically active substances from cultures of protozoa; the book reviews
thehistorical backgroundinconnectionwithcontemporarydevelopmentofthefield.
N.N.Sukhareva
ACKNOWLEDGMENTS
The research was performed infruitful cooperation with myresearch associates (V.
Urinyuk, T. Titiova, L. Udalova, R. Zeleneva, V. Brusovanik, M. Zaretskaya),
postgraduate students (N. Kalenik, M. Chuenkova, V. Vasilevskaya, V.
Khorokhorina), my colleagues at Moscow State University (Yu. Kozlov and I.
Makarenko), the colleagues from the Research Center of Antibiotics and
Chemotherapy (M. Vyadro, T. Terentjeva, I. Fornina and S. Navashin), L.
KazanskayafromtheFirstMoscowInstituteofMedicine;M.Levachev,S.Kulakova
and F. Medvedev from the Institute of Nutrition as well as L. Dyakonov from the
Research Institute on experimental veterinary. I appreciate the efforts of my son
SergeiSukharev.
I wouldliketopaytributetomyformer bossProfessor A.Silaev (formerChief
of the Laboratory of Antibiotics) who opened for me the way for creative work in
physiology of protozoa, biochemistry of lipids and microbial technology. Many
thanks to Professor N. Egorov (former Head of Microbiology Department) for his
priceless support during my scientific career. I am grateful to Professor Yu.
Poljansky,(the formerPresident ofAll-Union SocietyofProtozoologists) andDrT.
Beyer(ScientificSecretaryof theSociety)whohelpedmetoprepareandpublishthe
book "TheProtozoa asnewsubjects of biotechnology" (1989) at NaukaPublishers
oftheAcademyofSciencesinLeningrad.
The author thanks Mrs. Jennifer Jadin (Department of Biology, University of
-Maryland,USA)forherhelpinthemanuscriptediting.
INTRODUCTION
The large taxon known as the Protozoa contains a huge diversity of eukaryotic
species, comprised mainly of unicellular organisms. These microscopic creatures
represent a unique level of organization: they represent both a cell, highly
differentiated morphologically, and a whole organism, highly differentiated
functionally (Poljansky, 1978; Vickerman and Coombs, 1999). Many authors have
discussedthedefinitionoftheseorganismsandtheirlocationintheweboflife(Jahn
andBovee, 1967;Corliss, 1974, 1984;Krylovet al., 1980;Seravin, 1980;Leeet aI.,
1985).Onedefinition, asgivenbyLevin's committee:"TheProtozoa areessentially
single-celledeukaryoticorganisms.Theyarenotanaturalgroup,buttheyareplaced
together for convenience...The Protozoa may be considered a subkingdom of the
kingdom Protista.Ifthe classical classification is preferred, the Protozoa might be
consideredasubkingdomofthekingdomAnimalia" (LevineetaI.,1980).
The majority of the Protozoa are free-living organisms; they are distributed
elsewhere in environment, especially in soil and water. Among the Protozoa there
are several genera such as Trypanosoma, Leishmania, Plasmodium, Babesia,
Toxoplasma,Entamoebaetc, whichcause devastating diseases inhumans,domestic
andwildanimals,lowervertebrates,andinvertebrates,aswellasinplantsoftropical
and subtropical regions of the world (Hoare, 1972; Vickerman, 1985). It is not
possible to eradicate anyof these diseases by campaigns based on a single strategy
(Hirstand Stapley,2000). Onlyamultilateral approachcanbe helpful.Towards this
goal, new directions have been added to the traditional fields of protozoology: 1)
physiology and biochemistry of protozoa (1920's-1980's); 2) molecular biology,
genetics,biochemistryincludingenzymology,biophysics,andimmunology (1960's
present).
Scientists of the worldhave achieved manygoals infundamental studies of the
Protozoa during the 20th century and the data has been analyzed in numerous
reviews:
I.These organismshaveanamazingabilitytomodifytheirformsandfunctions
to adapt to diverse environments. Parasites have complex life cycles with various
modes of parasitism (Hoare, 1972; Soprunov, 1981; Coombs et aI., 1998).
Obtaining the complete developmental cycle of Leishmania mexicana in axenic
cultureshasbeenaremarkableachievement(Bates, 1994).
xii INTRODUCTION
2. Flagellates, which include parasites, trypanosomes and leishmanias,
taxonomically belong to class Zoomastigophorea, order Kinetoplastida, suborder
Trypanosomatina, family Trypanosomatidae (Lee et al, 1985). Important findings
onthese organisms werepublished:
a) Evolutionarily they display the first example of cytoplasmic DNA, the
kinetoplast, which was found as a massed single mitochondrion. This organelle
enables parasites to adapt to various energy sources and levels of available oxygen
(Leeetal., 1985;Vickerman andCoombs, 1999);
b) The kinetoplast consists of maxi- and minicircles of DNA. Interestingly,
dyskinetoplastic mutantshavingmajor maxicircles cannotbetransmitted (Englund et
al., 1982);
c) The kinetoplastid flagellates were the first organisms in which the
phenomenon ofRNAeditingwasdiscovered (Hideetal., 1997).
3. The diversity of the protozoan genome is truly fascinating. For example,
dinoflagellates lack histones but still possess typical eukaryotic cell organization.
They have extranuclear spindles that segregate into daughter chromosomes. The
Ciliophora (Ciliates) are unique in nuclear dimorphism: the diploid micronucleus is
usually nontranscriptive but divides by mitosis; it produces gamete nuclei after
mitosis during sexual processes. Conversely, the polygenomic macronucleus is
transcriptive butitdividesbyamitotic mode (Vickerman andCoombs etal., 1999).
4. The ciliate protozoa Paramecium aurelia was thefirst organism that allowed
scientists to raise the question of relationship between bacterial endosymbionts and
organelles ineukaryotes (Vickerman andCoombs,1999).
5. Glycosomes werediscovered to be intracellularmicrobodies containing most
of theenzymes ofglycolytic pathway inTrypanosoma brucei (Opperdoes and Borst,
1977;VisserandOpperdoes, 1980).
6. There were studied main types of protein glycosylation in flagellates: N
glycosylation and O-glycosylation (Parodi, 1993; Hounsell et al., 1996).
Glycoproteins such as variant surface glycoproteins (VSGs) contain N-linked
oligosacchrides. Protein N-glycosylation in trypanosomatids has unique features
(Parodi, 1993;de Lederkremerand Colli, 1995).Protein O-glycosylationtakes place
for biosynthesis of mucins and mucin-like glycoproteins (Hounsell et al., 1996).
Phosphoglycosylationhasbeendiscovered relatively recently (Haynes, 1997).
7.Trypanosoma brucei (bloodstream stage) is covered with VSGs (Vickerman,
1969;Cross, 1975;Cross, 1984;Turner, 1982;Englund etal., 1982;Paysand Nolan,
1998). VSGs possess antigenic properties (Cross, 1977; Borst and Cross, 1983;
Turner, 1984). By changing surface antigens trypanosomatids avoid host immune
response (Vickerman, 1969; Cross, 1977; 1984; Turner, 1982; 1984). This
phenomenon of antigen variation is considered to be the result of two different
processes: the alternative activation of VSGs expression sites and frequent DNA
rearrangements (Cross, 1975;Borst and Cross 1982;Turner, 1984; Pays and Nolan,
1998).Procyclin is the major surface protein of T. brucei procyclic forms (Pays and
Nolan, 1998).The other surface glycoproteins mayserveas receptors for toxins,
growth factors and membrane-bound enzymes such as glycosyltransferases and
glycosidases (PaysandNolan,1998).
INTRODUCTION Xlll
8.Glycosyl-phosphatidylinositols (GPIs) anchoring VSGs to the cell surface
were identified biochemically and functionally (de Lederkremer et al., 1976;
Ferguson et al., 1985a; Turco et al., 1987; McConville et al., 1990; McConville,
1991; Ferguson, 1999; Andrews, 2000). They are moieties of multifunctional
molecules, GPI-VSGs. By anchoring VSGs they create a macromolecularlayer that
protects receptors and transporters of parasites from the immune attack ofthe host
(Borst and Fairlamb, 1998).There were found anchores similar to GPI but linked
substances ofnon-protein nature (Turco, 1984;Ferguson etal., 1991;MeConville et
al., 1992;Ferguson, 1997;Descoteaux and Turco, 1999;Guha-Niyogi et al., 2001).
The diversity ofbiological functions ofglycosylatedlipidsmustbeemphasized: they
prevent agglutination, as well as complement- or hydrolytic enzyme-mediated cell
Iysis;they promote intracellular trafficking and intercellular transport (Ferguson,
1999); they regulate Leishmania susceptibility to insulin (Low and Saltie!, 1988),
they increase the parasite infectivity «McConville, 1991), and Ca2+ intracellular
concentration (Descoteaux and Turco, 1999); they take part in life cycle
differentiation ofKinetoplastida (Faria-e-Silvaetal., 1999).
9. The role of cytokines, eicosanoids and nitric oxide as effector molecules
against parasitic protozoa was reviewed (James, 1995; Liew et al., 1997;
Abrahamsohn, 1998;DaugschiesandJoachim, 2000;Brunet,2001)
10. Molecular and biochemical mechanisms and subsequent new therapeutic
approaches to the treatment of African trypanosomiasis have been summarized
(Wang, 1995; Urbina, 1997; Ferguson, 2000). For example, the attempt to replace
myristate by its close analogue, 10-(propoxy) decanoic acid in GPI anchor of
Trypanosoma brucei resulted in immense morphological changes in the
trypanosomes and their death within a few hours (Wang, 1995). Moreover
transporters involved inthetranslocation ofavarietyofmolecules acrossmembranes
are studied for their application in delivery of therapeutics into target cells
(Wiedlocha, 1998;Torres etal., 1999;Ferguson, 2000).
At present time, much research is aimed at the study of key-enzymes such as
phospholipases, cyclooxygenases, nitricoxide synthase, proteases andtheirproducts;
their gene expression and mechanisms of regulation; and, at their receptors,
stimulators and inhibitors (Fukuto and Chaudhuri, 1995; Kovac and Csaba, 1997;
Brunet, 2001; Das et al., 2001; Nie and Honn, 2002). The study of macrophage
receptors for variousbiologically active substances isthecurrent topicbecause these
immunocompetentcellsrepresent first lineof defense inmammals against infectious
agents and tumors (Makarenko et aI., 1988; Sukhareva, 1989; Paulnock and Coller,
200I; Almeida and Gazzinelli, 200I; Bishop-Bailey et al., 2002). Some
Mastigophora (Flagellates) possess metabolic dualism and are capable of producing
substances characteristic of animals and/or plants depending on habitat or conditions
of cultivation. The combinatorial biochemistry of nature is really complicated
(Verdine, 1996). These substances do not occur in prokaryotes, which are
traditional producersofantibiotics andotherbiologically activesubstances.
The representatives of the Protozoa are gradually acquiring their place as subjects
ofmicrobial technology forproduction ofbiologicallyactivesubstances.