pubmed_result Michael de Graaff

1. Arch Virol. 2001;146(3):571-88.
Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral
RNA polymerase. 3'-terminal non-coded guanosine and coat protein are insufficient
factors for full-size plus-strand synthesis.
Houwing CJ(1), Huis in 't Veld M, Zuidema D, de Graaff M, Jaspars EM.
Author information:
(1)Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University,
The Netherlands.
Replication complexes of alfalfa mosaic virus produce in vivo large quantities of
plus-strand RNAs, but this production is fully dependent on the presence of coat
protein. In order to study this process of RNA-dependent and coat
protein-regulated RNA synthesis we have isolated the three natural minus-strand
RNAs (containing any posttranscriptional modification that might have occurred)
and have tested them for coat protein binding sites and template activity in an
in vitro system with the viral RNA polymerase. The enzyme was prepared by an
advanced isolation procedure. All three minus strands had a single non-coded G at
their 3' terminus. They were not able to withdraw coat protein subunits from
virions as free virion RNAs do. No sites protected by coat protein against
ribonuclease T1 degradation were found. Two large T1 oligonucleotides from minus
RNA 1 and one from minus RNA 3 were bound by coat protein to Millipore filters.
Except for minus RNA 3 which caused a minute amount of full-size plus strand to
be synthesized, the minus strands did not function as templates for full-size
complementary strands. On the other hand, they gave rise to a number of
well-defined shorter products, the synthesis of which was stimulated by the
addition of coat protein. These products could not be elongated by a chase
treatment and were probably the result of internal initiations. It is concluded
that, although posttranscriptional modifications of the template and the presence
of coat protein may be necessary factors for plus-strand RNA synthesis, they are
certainly not sufficient. Our purified in vitro system needs further
sophistication.
PMID: 11338391 [PubMed - indexed for MEDLINE]
2. Anticancer Drugs. 1999 Feb;10(2):213-8.
In vitro antagonistic cytotoxic interactions between platinum drugs and taxanes
on bone marrow progenitor cell CFU-GM.
de Graaff M(1), Maliepaard M, Pluim D, Floot BJ, Slaper-Cortenbach IC, Schellens
JH.
Author information:
(1)Department of Experimental Therapy and Medical Oncology, The Netherlands Cancer

Institute, Amsterdam.
We have designed and used an in vitro bone marrow cell culturing system for
investigating pharmacodynamic interactions between platinum anti-cancer drugs and
taxanes. With this system, in which the bone marrow progenitor cell CFU-GM is
proliferating and differentiating into granulocytes and monocytes, we could show
a strong antagonistic cytotoxicity of the combination carboplatin and Taxotere,
in three different schedules, and of the combination cisplatin and Taxol, in two
out of the three schedules tested. Modulation of intracellular platinum drug
accumulation in granulocytes and monocytes does not seem to be a plausible
explanation for the observed antagonism. In vitro co-incubation of
granulocytes/monocytes with the combination carboplatin and Taxotere did not
reveal an effect of Taxotere on intracellular platinum accumulation. Although
Taxol reduced intracellular cisplatin levels by 12%, this effect was not
significantly different from the co-incubation of cisplatin with Cremophor EL,
the solvent for paclitaxel in Taxol. The toxicity data obtained in this study
seem to be in accordance with recent clinical trials where combination therapies
with platinum drugs and taxanes resulted in marked reductions in myelosuppression
in patients. Therefore, these types of assays could be useful as to the
assessment of bone marrow toxicities of clinically important drug combinations.
3. J Biol Chem. 1997 Jul 4;272(27):16815-21.
Interactions between the human RNA polymerase II subunits.
Acker J(1), de Graaff M, Cheynel I, Khazak V, Kedinger C, Vigneron M.
Author information:
(1)Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS/INSERM/ULP),
F-67404 Illkirch Cedex C.U. de Strasbourg, France.
As an initial approach to characterizing the molecular structure of the human RNA
polymerase II (hRPB), we systematically investigated the protein-protein contacts
that the subunits of this enzyme may establish with each other. To this end, we
applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect
cells, which were coinfected with all possible combinations of recombinant
baculoviruses expressing hRPB subunits, either as untagged polypeptides or as
glutathione S-transferase fusion proteins. This is the first comprehensive study
of interactions between eukaryotic RNA polymerase subunits; among the 116
combinations of hRPB subunits tested, 56 showed significant to strong
interactions, whereas 60 were negative. Within the intricate network of
interactions, subunits hRPB3 and hRPB5 play a central role in polymerase
organization. These subunits, which are able to homodimerize and to interact, may
constitute the nucleation center for polymerase assembly, by providing a large
interface to most of the other subunits.

4. Virology. 1995 Nov 10;213(2):650-4.
Interaction between RNA-dependent RNA polymerase of alfalfa mosaic virus and its
template: oxidation of vicinal hydroxyl groups blocks in vitro RNA synthesis.
De Graaff M(1), Thorburn C, Jaspars EM.
Author information:
The Netherlands.
In the life cycle of a (+)-strand RNA plant virus the processes of template RNA
recognition and initiation of the synthesis of a complementary strand by the
viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a
template-dependent in vitro RNA synthesizing system of alfalfa mosaic virus
(AIMV) we were able to study the effect of small chemical modifications of the 3'
end of the template RNAs on product formation. After oxidation of the 3'-terminal
nucleoside of the template no products could be detected. Presumably, RNA
synthesis was blocked at the stage of initiation, since the promoter of the RdRp
is internal (A. C. Van der Kuyl et al., Virology 176, 346-354, 1990). Blocking
was probably due to an irreversible binding of the enzyme to the 3' end of the
modified RNA. Using this system it was shown that in template competition
experiments the RdRp of AIMV displays a high specificity for its cognate
template, either before or after the oxidation of the 3'-terminal nucleoside.
From this it was concluded that periodate modification of the 3'-terminal
nucleoside has little or no effect on template recognition. Furthermore, we
showed that the viral coat protein, which forms a part of the viral polymerase
(R. Quadt et al., Virology 182, 309-315, 1991), was not the main target involved
in the inhibition of RNA synthesis.
5. FEBS Lett. 1995 Sep 11;371(3):219-22.
RNA duplex unwinding activity of alfalfa mosaic virus RNA-dependent RNA
polymerase.
de Graaff M(1), Houwing CJ, Lukács N, Jaspars EM.
Author information:
(1)Institute of Molecular Plant Sciences, Leiden University, The Netherlands.
An RNA-dependent RNA polymerase (RdRp) purified from alfalfa mosaic
virus-infected tobacco is capable of synthesizing in vitro full-size RNAs of
minus and plus polarities. However, the enzyme is not able to perform a complete

replication cycle in vitro. The products were found to be completely base-paired
to their templates. The enzyme was able to use double-stranded RNA as a template
for RNA synthesis if it could initiate from a single-stranded promoter. The
inability (of most) of our enzyme preparations to create a single-stranded
initiation site could explain why they could not perform a complete replication
cycle in vitro. This is the first report on duplex RNA unwinding activities by a
plant viral RdRp.
6. Virology. 1995 Apr 20;208(2):583-9.
In vitro evidence that the coat protein of alfalfa mosaic virus plays a direct
role in the regulation of plus and minus RNA synthesis: implications for the life
cycle of alfalfa mosaic virus.
De Graaff M(1), Man in't Veld MR, Jaspars EM.
Author information:
(1)Virology Section, Gorlaeus Laboratories, Leiden University, The Netherlands.
The coat protein of alfalfa mosaic virus has both structural and regulating
functions. The latter is evident from the fact that the genomic RNAs of the
virus, although they are of messenger polarity, cannot start an infection cycle
in the absence of cost protein. The reason could be that the coat protein is
needed for viral RNA synthesis. Indeed, the coat protein has been found in tight
association with the viral RNA polymerase (R. Quadt et al., 1991, Virology 182,
309-315). To investigate the role of the coat protein, if any, in viral RNA
synthesis, we have isolated that viral RNA polymerase (RNA-dependent RNA
polymerase, RdRp) from mock-inoculated tobacco plants transformed with cDNAs 1
and 2, known as P12 plants (P. E. M. Taschner et al., 1991, Virology 181,
687-693), which express the nonstructural proteins P1 and P2. Such an enzyme
(called M-RdRp) will contain the viral subunits P1 and P2 but not the coat
protein. As a comparison we also isolated the RdRp from virion-inoculated P12
plants (C-RdRp). This enzyme will contain the coat protein. We found that both
M-RdRp and C-RdRp could synthesize minus RNA, showing that coat protein is not
needed for minus-strand synthesis. In contrast, minus-strand synthesis by both
enzymes was inhibited by coat protein. Plus-strand synthesis was unaffected by
coat protein in the case of C-RdRp, but strongly stimulated by coat protein in
the case of M-RdRp. These data might explain why infected cells, which do not
produce coat protein, display a very low accumulation of viral plus-strand RNA.
They also give a possible explanation for the noninfectious character of the
genomes of alfalfa mosaic virus and ilarviruses in the absence of coat protein.
The fact that an active enzyme could be isolated from the same membrane fraction
in infected and noninfected P12 plants shows that coat protein is not needed for
assembly and targeting of the viral RNA polymerase.

7. Virology. 1993 Jun;194(2):878-81.
Localization and biochemical characterization of alfalfa mosaic virus replication
complexes.
De Graaff M(1), Coscoy L, Jaspars EM.
Author information:
The Netherlands.
Replication complexes were isolated from alfalfa mosaic virus-infected tobacco
protoplasts. Most of the RNA-synthesizing activity appears to colocalize with the
intact chloroplasts upon sucrose-gradient centrifugation of cellular homogenates.
Further analysis of these replication complexes showed that the enzyme is
strongly associated with the outside of the chloroplasts, the endogenous template
being well protected against ribonuclease action. RNA polymerase activity is
sensitive to protease treatment of intact chloroplast fraction showing that an
essential part of the enzyme complex is facing the in vitro medium, and probably
the cytosol in vivo.
8. Eur J Immunol. 1992 Jul;22(7):1851-6.
Induction of homotypic T cell adhesion by triggering of leukocyte
function-associated antigen-1 alpha (CD11a): differential effects on resting and
activated T cells.
Koopman G(1), de Graaff M, Huysmans AC, Meijer CJ, Pals ST.
Author information:
(1)Department of Pathology, University of Amsterdam, The Netherlands.
The leukocyte integrin LFA-1 (CD11a/CD18) plays a key role in many adhesive
interactions involving cells of the immune system. Recently, it has been shown
that LFA-1 is not only involved in cell adhesion, but that stimulation of LFA-1
can also contribute to cell activation. We now demonstrate that triggering of
LFA-1 on T lymphocytes by monoclonal antibodies (mAb) against the LFA-1 alpha
chain, but not against the LFA-1 beta chain, promotes cell adhesion. Induction of
homotypic adhesion was only observed in T cells that had been pre-activated with
anti-CD3 and not in resting peripheral blood T lymphocytes. The induced homotypic
adhesion is mediated by LFA-itself, because it was inhibited by anti-LFA-1 beta
mAb. This notion is supported by the temperature and divalent cation dependence
which is characteristic of LFA-1-mediated adhesion. mAb against ICAM-1 (CD54) did
not block LFA-1 alpha-induced adhesion. The sensitivity of LFA-1 alpha-induced

adhesion to H7, which prevents the activation of protein kinase C and protein
kinase A, and to cytochalasin B, which inhibits microfilament formation, suggests
that the activation of the LFA-1 pathway through the LFA-1 alpha chain involves
cell activation and requires an intact cytoskeleton.
9. J Immunol. 1990 Dec 1;145(11):3589-93.
Triggering of the CD44 antigen on T lymphocytes promotes T cell adhesion through
the LFA-1 pathway.
Koopman G(1), van Kooyk Y, de Graaff M, Meyer CJ, Figdor CG, Pals ST.
Author information:
(1)Department of Pathology, Free University, Amsterdam, The Netherlands.
The CD44 molecule, a molecule which has been previously known as Hermes, Pgp-1,
extracellular matrix receptor III, and In(Lu)-related p80, is currently thought
to be involved in several steps of normal immune cell function, including
lymphocyte adhesion to high endothelial venules and to the extracellular matrix
and T cell activation. We now demonstrate that triggering of CD44 on T
lymphocytes by anti-CD44 mAb promotes cell adhesion. The induced homotypic
adhesion is mediated by lymphocyte function-associated antigen-1 (LFA-1), because
it was inhibited by anti-LFA-1 antibodies and not by anti-LFA-3 antibodies. This
notion is supported by the temperature and Mg2+ dependence which is
characteristic of LFA-1-mediated adhesion. Moreover, the sensitivity of
CD44-induced adhesion to AMG and H7, which both prevent the activation of protein
kinase C, and to cytochalasin B, which inhibits microfilament formation, suggests
that the activation of the LFA-1 pathway via CD44 involves protein kinase C
activation and requires an intact cytoskeleton.
10. Annual Review of Phytopathology Vol. 32: 311-335
(Volume publication date September 1994)
Plant Viral RNA Synthesis in Cell-Free Systems
M de Graaff, and E M J Jaspars
Author information:
The Netherlands.

pubmed_result Michael de Graaff

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