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M Smahel

Publications and source records attributed to M Smahel.

25 records · Page 2Linked to original sources
PubMed1997

Putative regulatory sequence in human papillomavirus type 16 E2 open reading frame.

A 114 bp fragment of the human papillomavirus type 16 (HPV16) E2 open reading frame (nt. 3142-3255) containing a putative estrogen responsive element (ERE) was amplified and cloned into pBLCAT2 plasmid in both sense (p159-4) and anti-sense (p164) orientation. The plasmids were transfected into human breast-cancer cell line MCF-7 containing estrogen receptor and the cultures were kept in the presence or absence of beta-estradiol. The chloramphenicol acetyltransferase (CAT) activity was not influenced by estrogen. However, a silencer effect was observed both in cultures transfected with p159-4 and p164 plasmids. We prepared and cloned synthetic fragments containing the putative ERE and failed to prove that the palindrome in the putative ERE was responsible for the silencer activity.

Base Sequence↗
PubMed1993

Characterization of ion channels from Acetabularia plasma membrane in planar lipid bilayers.

Plasma membrane from Acetabularia acetabulum was prepared by aqueous-polymer two-phase partitioning and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers by stirring in the presence of a (cis:trans) 325:100 mM KCl gradient. Under these conditions five distinct K(+)-selective channels were observed which had unitary chord-conductances (determined between 30 mV either side of the reversal potential) and frequencies of incorporation (in parentheses) of 1,600 pS (26%), 485 pS (21%), 259 pS (53%), 140 pS (37%) and 27 pS (37%). Two Cl(-)-selective channels were also observed, which had unitary chord-conductances of 8 and 48 pS and were present in 21 and 16% of bilayers, respectively. The voltage dependencies of channel open probability (Po), open-state time constant (tau o) and closed-state time constant (tau c) were determined for the 259, 140 and 27 pS K+ channels. The Po of all three channels increased with increasingly positive membrane potentials. Thus, since these channels were oriented with their extracellular face adjacent to the cis chamber, which was grounded, all would exhibit outward rectification in vivo. Changes in Po were effected by modulation of tau c in all channels, which shortened as membrane potentials became more positive, and also of tau o in the 140 and 27 pS channels, which increased as membrane potentials became more positive. Extracellular (cis) KCl concentration (and/or the KCl gradient across the bilayer) affected the Po of all three K+ channels, shifting the Po/membrane potential relationship in the direction of the change in the potassium reversal potential. In all channels this was achieved largely by changes in tau c.

Acetabularia↗
PubMedDate not supplied

DNA vaccine against oncogenic hamster cells transformed by HPV16 E6/E7 oncogenes and the activated ras oncogene.

The capability of DNA to elicit anti-tumour immunity was studied using human papillomavirus type 16 (HPV16)-transformed Syrian hamster cells denoted K3/II. These cells had been derived after cotransfection of primary kidney cell cultures with p16HHMo plasmid containing E6/E7 oncogenes of HPV16 and pEJ6.6 plasmid containing the activated human H-ras oncogene; they express both the HPV16 and activated H-ras genes. As a DNA vaccine, the p16HHMo plasmid was used. Three doses of the plasmid (either 100 microg or 10-15 microg per dose) were administered intramuscularly at 3-week intervals. The animals were challenged with four different doses (10(3)-10(6) per animal) of K3/II cells 10 days after the last plasmid injection. In one experiment the lower dose of plasmid DNA was also given in a mixture with the cationic lipid DOTAP. In another experiment, the pEJ6.6 plasmid (100 microg per dose) was used either alone or in combination with p16HHMo. In all experiments animals inoculated with the same doses of pBR322 plasmid served as controls. A moderate protective effect was observed in animals inoculated with the 100-microg doses of p16HHMo, but not in those inoculated with 10-15 microg of the same plasmid, whether given with or without DOTAP. A protective effect was also observed after administration of the pEJ6. 6 plasmid. At the time of challenge a portion of the p16HHMo-immunized, but not the pBR322-treated, animals possessed antibodies reactive in ELISA with peptides derived from the N-terminal portion of HPV16 E7 protein and with one peptide derived from E6 protein, while two other E6 peptides exhibited non-specific reactivity.

Animals↗
PubMedDate not supplied

Chemoimmunotherapy of cancer: potentiated effectiveness of granulocyte-macrophage colony-stimulating factor and ifosfamide derivative CBM-4A.

The effectiveness of combined chemoimmunotherapy with ifosfamide derivative CBM-4A and granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated in two experimental tumor models, 3MC-induced MHC class I+ sarcoma Mc12 and HPV16 E6/E7 oncogene-induced MHC class I- carcinoma MK16, transplanted in syngeneic mice. Treatment of Mc12 and MK16 tumor-bearing mice with GM-CSF or CBM-4A alone produced moderate anti-tumor effects. However, when the tumor-bearing mice were first treated i.p. with a single dose of CBM-4A (150 mg/kg) and three days later peritumorally with five daily doses of GM-CSF (100 ng/day), substantially stronger tumor-inhibitory effects were observed. The results indicate that in both, MHC class I+ and MHC class I- tumors, the combined chemoimmunotherapy can inhibit tumor progression more effectively than GM-CSF therapy or chemotherapy alone, and they suggest that GM-CSF should be considered as adjuvant to chemotherapy in clinical trials with HPV 16-associated neoplasms.

Animals↗
PubMedDate not supplied

Properties of Syrian hamster cells transformed by human papillomavirus type 16.

Adult Syrian hamster kidney cells were transfected with a mixture of plasmids containing human papillomavirus type 16 (HPV16) E6/E7 open reading frames (ORFs), activated Ha-ras gene and neomycin resistance gene. From these cultures two lines were isolated which were oncogenic for newborn and 5-day-old but not for 3-week-old hamsters. Sublines oncogenic for 3-7-week-old hamsters were derived from tumours formed in animals inoculated within 5 days of birth. The cells contained HPV 16 DNA in an integrated form and HPV16 transcripts. The transcript patterns in low and high oncogenicity sublines were different. Very few tumour-bearing animals possessed antibodies reactive with E6- and E7-derived synthetic peptides. On the other hand a majority of these animals gave a positive reaction in the lymphoproliferation assay with either E6 and E7 peptides or extracts from the transformed cells.

Animals↗