Volume 49 (2003) No. 6

Original Articles
The Use of Housekeeping Genes (HKG) as an Internal Control for the Detection of Gene Expression by Quantitative
Real-Time RT-PCR
V. ULLMANOVÁ, C. HAŠKOVEC

Quantitative real-time RT-PCR is a very useful technique for estimating gene expression at the mRNA level. The expression of a tested
gene has to be compared with that of a control gene. Various housekeeping genes have been used as control genes in different systems.
 In our study we tested several housekeeping genes in the model of gene expression after induction of apoptosis and differentiation.
The myeloid cell lines were incubated with phorbol esters, butyric acid and combination of TNFα and IFNγ to induce differentiation. Camptothecin was used for induction of apoptosis. Tested control genes included β-2 microglobulin, GAPDH, 18S ribosomal RNA and abl. GAPDH was found to be the best control gene in the apoptotic system. Different control genes were suitable for different systems where differentiation or senescence was induced. Our results show that attention should be paid to the choice of an appropriate control gene of quantitative real-time RT-PCR for different experimental models and various experimental conditions.
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Adjuvant Cytokine Treatment of Minimal Residual Disease after Surgical Therapy in Mice Carrying HPV16-Associated
Tumours: Cytolytic Activity  of Spleen Cells  from Tumour Regressors
M. INDROVÁ, R. MIKYŠKOVÁ, T. JANDLOVÁ, V. VONKA, J. BUBENÍK,
J. BIEBLOVÁ

Identification of the HLA Alleles with Low and No Cell Surface Expression in the Czech Population
N. BENDUKIDZE, E. IVAŠKOVÁ,  L. ZAHLAVOVÁ, A. SLAVCEV, L. KUPKOVÁ, H. SAJDLOVÁ,
S. DAY, P. P. J. DUNN

The presence of the A*24020102L allele was implicated in one donor from the Czech Bone Marrow Donor Registry (CBMD), who serologically was type  A2; B44, B55; Cw1, Cw7. The DRB4*01030102N allele was identified in one healthy donor and in one patient with MDS during routine HLA class II DNA typing.
The DRB4*01030102N allele was identified in the patient’s father, who had CML, and was associated with the HLA-A3-B7-Cw7-DRB1*0701-DQB1*0303 haplotype, which is common for European populations.
In order to avoid mistyping, both techniques, serology and molecular biology must be used for HLA typing, especially for cases where just
one antigen appeared to be present using serological methods.
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Short Communications
MHC Class I⁺ and Class I^- HPV16-Associated Tumours Expressing E7 Oncoprotein Do Not Cross-react
in Immunization/Challenge Experiments
J. ŠÍMOVÁ, R. MIKYŠKOVÁ, V. VONKA, J. BIEBLOVÁ, J. BUBENÍK, T. JANDLOVÁ

It has been demonstrated repeatedly that a high proportion of tumours derived from MHC class I⁺ precursors are MHC class I^-.
Since a major task in immunotherapy strategies for treatment of malignancies is to develop polyvalent tumour vaccines efficient against a
 broad spectrum of tumours, we have examined whether MHC class I⁺ cell-based tumour vaccines can cross-protect against homologous
 MHC class I^- tumour challenge and vice versa. For these purposes, we have used two oncogenic cell lines induced independently by co?transfection of murine H-2^b cells with E6/E7 HPV16 and activated Ha-ras oncogenes, the tumours TC-1 (MHC class I+, HPV16 E7⁺)
 and MK16/1/IIIABC (MHC class I^-, HPV16 E7⁺). Surprisingly, it was found that these two tumours do not cross-react, although both
of them contain the crucial HPV16-coded tumour rejection antigen E7. Preimmunization with the MHC class I⁺ tumour did not protect
 against a subsequent challenge with the MHC class I^- tumour and vice versa; immunization with the TC-1 tumour could protect syngeneic
 mice against the TC-1 tumour challenge and, similarly, immunization with the MK16/1/IIIABC tumour could protect mice against the MK16/1/IIIABC tumour challenge. If this finding can also be confirmed as a more general phenomenon with other MHC class I⁺ and class I^- tumours, it could have serious implications for design of immunotherapeutic vaccines and protocols.
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No Association between Oxidative Stress and IL-6 in NIDDM Patients with Nephropathy
Ş. ALSANCAK, M. AYDIN, A. YILDIZ, S. SALMAN, Ş. SEÇKIN

Oxidative stress is hypothesized to play a role in the development of diabetes with and without nephropathy. In addition, it has been suggested that some metabolic abormalities associated with diabetes may be due to cytokine overproduction. In the light of this knowledge, we aimed to measure MDA levels as a marker of oxidative stress and the IL-6 level in diabetes with and without different stages of nephropathy. Plasma MDA levels  in the group of NIDDM patients with advanced nephropathy were significantly higher than the group of NIDDM patients
without nephropathy, which was significantly higher compared with the control group. Although IL-6 levels were elevated in diabetic
groups with and without nephropathy in comparison with the control, no significant difference was found between patient groups. As a conclusion, oxidative stress may play an important role in diabetes with and without nephropathy.
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Monoclonal Antibody Register
Monoclonal Antibody BF-06 against the Heavy Chain of Clathrin
L. MACŮREK, M. ZÍKOVÁ, E. DRÁBEROVÁ, P. DRÁBER

Clathrin is a ubiquitously expressed protein which polymerizes into a coat-like lattice on the cytoplasmic surface of cellular membranes
 (Brodsky et al., 1988). Formation of a polyhedral lattice is enabled by self-assembly of clathrin triskelions, which are composed of three
 copies of clathrin heavy chain (~180kDa) and three light chain (~30kDa) subunits. Clathrin is a major component of coated vesicles
 (Pearse et al., 1975), from which it dissociates at higher ionic strength or at basic pH (Liu et al., 1995). Reassociation of purified clathrin
 in vitro occurs after lowering the pH. Clathrin polymerization results in membrane invagination and is dynamically regulated by light
 chains in vivo. Clathrin-coated vesicles are responsible for both the receptor-mediated endocytosis and the secretory vesicle budding
 from the trans-Golgi network. Interaction of clathrin with the membrane is indirect and is mediated by adaptor proteins (AP 1–4)
 specific for different cellular compartments (Kirchhausen, 1999). Immunofluorescence with anti-clathrin antibodies results in a punctuate
 staining of the vesicles in the perinuclear region of interphase cells. In mammalian cells, localization of clathrin on microtubular arrays
of mitotic spindles was also reported (Okamoto et al. 2000).
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