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Biomedical subjects

M Kubista

Publications and source records attributed to M Kubista.

At least 19 recordsLinked to original sources
PubMed2003

Classification of commercial apple beverages using a minimum set of mid-IR wavenumbers selected by Procrustes rotation.

When infrared spectral data are used in classification and/or multivariate regression methods there can be problems related to both chemical understanding and computation speed due to the large number of wavenumbers in each spectrum. Here, it is shown that the Procrustes rotation technique can be used to select a minimum set of spectral variables (wavenumbers) to perform classification and regression. Procrustes rotation was coupled to several multivariate methods as PLS, SIMCA and potential curves (a maximum likelihood classification method). The practical problem of implementing a screening methodology for classifying apple juice-based beverages according to their contents of "pure" apple juice was addressed using attenuated total reflectance, mid-IR spectroscopy. It is found that two of the original wavenumbers are almost as good predictors as all the 176 initial ones.

Autoanalysis↗
PubMed2002

Interleukin-1 system and sex steroid receptor gene expression in human endometrial cancer.

OBJECTIVES: The interleukin-1 system is known to play a pivotal role in human physiology and reproduction. In the cycling endometrium, interleukin-1alpha activity is controlled by sex steroids and is confined to the perimenstrual phase, where it is involved in the events leading to tissue lysis and menstruation. Since local tissue degradation is also a feature of malignant tumors, our goal was to analyze the gene expression of interleukin-1alpha and other interleukin-1 family members and compare it with estrogen receptor alpha, estrogen receptor beta, and progesterone receptor mRNA expression in 27 endometrial carcinomas and 13 normal endometria. METHODS: Endometrial tumor tissues were obtained during hysterectomy for endometrial cancer, and normal endometrium was sampled in women undergoing surgical procedures for nonendometrial pathologies. Gene expression was analyzed by reverse transcription polymerase chain reaction. Protein expression was detected and localized by immunohistochemical staining. RESULTS: A strong gene expression of interleukin-1 type I receptor, estrogen recptor alpha, and progesterone receptor was detected in all tumor tissues and in the majority of benign endometrial tissues. However, in contrast to nonmalignant endometria, variable amounts of interleukin-1beta and interleukin-1 receptor antagonist mRNA were also detected in most of the tumor samples. Gene expression of interleukin-1alpha and estrogen receptor beta was considerably less frequent, with interleukin-1alpha being absent in all peri- and postmenopausal endometria and in all but one of the well-differentiated tumors. With decreasing differentiation interleukin-1alpha gene expression became more frequent. In these cases, interleukin-1alpha protein was detected predominantly in epithelial tumor cells of lower-grade tumors. CONCLUSION: We have demonstrated the presence of the interleukin-1 system in endometrial malignancies, and found a negative correlation between interleukin-1alpha and tumor differentiation. We hypothesize that the nonphysiological expression of interleukin-1alpha in less differentiated tumors might contribute to their invasiveness and malignant behavior.

Carcinoma, Endometrioid↗
PubMed2001

Tuberous sclerosis causing mutants of the TSC2 gene product affect proliferation and p27 expression.

The autosomal dominant disease tuberous sclerosis (TSC) is caused by mutations in either TSC1 on chromosome 9q34, encoding hamartin, or TSC2 on chromosome 16p13.3, encoding tuberin. TSC is characterized by hamartomas that occur in many organs of affected patients and these have been considered to likely result from defects in proliferation control. Although the true biochemical functions of the two TSC proteins have not been clarified, a series of independent investigations demonstrated that modulated hamartin or tuberin expression cause deregulation of proliferation/cell cycle in human, rodent and Drosophila cells. In support of tuberin acting as a tumor suppressor, ectopic overexpression of TSC2 has been shown to decrease proliferation rates of mammalian cells. Furthermore, overexpression of TSC2 has been demonstrated to trigger upregulation of the cyclin-dependent kinase inhibitor p27. We report that three different naturally occurring and TSC causing mutations within the TSC2 gene eliminate neither the anti-proliferative capacity of tuberin nor tuberin's effects on p27 expression. For the first time these data provide strong evidence that deregulation of proliferation and/or upregulation of p27 are not likely to be the primary/only mechanisms of hamartoma development in TSC. These results demand reassessment of previous hypotheses of the pathogenesis of TSC.

Animals↗
PubMed2001

Free-probe fluorescence of light-up probes.

The fluorescence enhancement of light-up probes (thiazole orange (TO) conjugated peptide nucleic acids (PNAs)) upon hybridization to target nucleic acid depends on the probe sequence, mainly due to large variations in free-probe fluorescence. Here we study three probes where the fluorescence in free state varies more than 50-fold. We find that this variation is due to a fraction that has TO intramolecularly "back-bound" to the PNA bases. The intramolecular affinity constant for this unimolecular interaction was determined by temperature titrations using absorption spectroscopy, and the fluorescence quantum yields of the probes in back-bound conformation were calculated. The molar ratio of probes in back-bound conformation was 0.70-0.96 at 30 degrees C and 0.40-0.73 at 60 degrees C, and the fluorescence quantum yield in back-bound conformation varied between 0.0020 and 0.077 at 30 degrees C, and 0.00065-0.029 at 60 degrees C. These data show that the variation in free-probe fluorescence depends mainly on the fluorescence quantum yield of the probe in back-bound conformation and to a much lesser extent on the tendency of the probe to adopt the back-bound conformation. With increasing temperature the free-probe fluorescence decreases owing to both reduced degree of back-binding and a decrease of the fluorescence quantum yield in back-bound conformation.

Base Sequence↗
PubMed2001

Tuberous sclerosis gene products in proliferation control.

Two genes, TSC1 and TSC2, have been shown to be responsible for tuberous sclerosis (TSC). The detection of loss of heterozygosity of TSC1 or TSC2 in hamartomas, the growths characteristically occurring in TSC patients, suggested a tumor suppressor function for their gene products hamartin and tuberin. Studies analyzing ectopically modulated expression of TSC2 in human and rodent cells together with the finding that a homolog of TSC2 regulates the Drosophila cell cycle suggest that TSC is a disease of proliferation/cell cycle control. We discuss this question including very recent data obtained from analyzing mice expressing a modulated TSC2 transgene, and from studying the effects of deregulated TSC1 expression. Elucidation of the cellular functions of these proteins will form the basis of a better understanding of how mutations in these genes cause the disease and for the development of new therapeutic strategies.

Active Transport, Cell Nucleus↗
PubMed2000

Defective antigen presentation resulting from impaired expression of costimulatory molecules in breast cancer.

Previous experiments from our laboratory have shown that immune mechanisms aiming at the destruction of tumour cells including the recognition of target cells and their elimination via the expression of intercellular adhesion molecule-1 (ICAM-1; CD54), the production of tumour necrosis factor-alpha (TNF-alpha) by monocytes and appropriate function of lymphocyte subpopulations were defective in breast cancer. Previous observations were extended to assess expression levels and regulatory mechanisms of costimulatory molecules CD54, CD80 and CD86 on monocytes derived from patients with early breast cancer (EBC). In addition, antigen presentation by antigen-presenting cells (APC) was analyzed within this context. We report that monocytes derived from patients with EBC exhibited significantly decreased expression levels of CD54 (p = 0.0002), CD80 (p = 0.009) and CD 86 (p = 0.002) compared with monocytes derived from healthy females. Simultaneously, lipopolysaccharide (LPS)-induced TNF-alpha production of monocytes was found to be defective in patients with EBC. Finally, T-cell proliferation in response to tetanus toxoid (TT) was significantly decreased in patients with EBC compared with healthy control females (p < 0.0001). Furthermore, T-cell proliferation in response to TT-pulsed APC derived from healthy controls was significantly inhibited in the presence of anti-CD54 and/or anti-CD80 antibodies in a dose-dependent manner, thus corroborating the necessity of the presence of CD54 and CD80 as costimulatory molecules in the present setting. We conclude that monocytes derived from patients with EBC showed a simultaneous defect of expression of CD54 and its regulation via TNF-alpha, CD80 and CD86 as well as T-cell proliferation following exposure to TT-pulsed APC. Based upon these findings, it is speculated that defects in costimulatory molecule expression might contribute to tolerance of the immune system towards the presence of malignant cells in patients with EBC.

Adult↗
PubMed2000

Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution.

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.

Base Pair Mismatch↗
PubMed2000

Rapid and specific detection of PCR products using light-up probes.

Newly developed light-up probes offer an attractive tool for PCR product detection. The light-up probe, which consists of a thiazole orange derivative linked to a peptide nucleic acid oligomer, hybridizes specifically to complementary nucleic acids. Upon hybridization the thiazole orange moiety interacts with the nucleic acid bases and the probe becomes brightly fluorescent. This eliminates the need to separate bound from unbound probes and reduces the risk of cross contamination during sample handling. We demonstrate here the applicability of light-up probes in two different PCR assays, one directed towards the human beta-actin gene and the other towards the invA gene of Salmonella. The probes do not interfere with the PCR reaction and can either be included in the sample mixture or added after completed amplification. The specificity of the probe is found to be excellent: a single-base mismatch in the target sequence is sufficient to prevent probe binding as indicated by the lack of fluorescence increase. Furthermore, a clear correlation is found between the intensity of gel bands and the measured probe fluorescence in solution, which suggests that the amount of PCR products can be quantified using light-up probes.

Actins↗
PubMed1999

Nucleosome structural features and intrinsic properties of the TATAAACGCC repeat sequence.

Nucleosomes, the fundamental building blocks of chromatin, play an architectural role in ensuring the integrity of the genome and act as a regulator of transcription. Intrinsic properties of the underlying DNA sequence, such as flexibility and intrinsic bending, direct the formation of nucleosomes. We have earlier identified genomic nucleosome-positioning sequences with increased in vitro ability for nucleosome formation. One group of sequences bearing a 10-base pair consensus repeat sequence of TATAAACGCC had the highest reported nucleosome affinity from genomic material. Here, we report the intrinsic physical properties of this sequence and the structural details of the nucleosome it forms, as analyzed by footprinting techniques. The minor groove is buried toward the histone octamer at the AA steps and facing outwards at the CC steps. By cyclization kinetics, the overall helical repeat of the free DNA sequence was found to be 10.5 base pairs/turn. Our experiments also showed that this sequence is highly flexible, having a J-factor 25-fold higher than that of random sequence DNA. In addition, the data suggest that twist flexibility is an important determinant for translational nucleosome positioning, particularly over the dyad region.

Animals↗
PubMed1999

Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences.

Our laboratories recently completed SELEX experiments to isolate DNA sequences that most-strongly favor or disfavor nucleosome formation and positioning, from the entire mouse genome or from even more diverse pools of chemically synthetic random sequence DNA. Here we directly compare these selected natural and non-natural sequences. We find that the strongest natural positioning sequences have affinities for histone binding and nucleosome formation that are sixfold or more lower than those possessed by many of the selected non-natural sequences. We conclude that even the highest-affinity sequence regions of eukaryotic genomes are not evolved for the highest affinity or nucleosome positioning power. Fourier transform calculations on the selected natural sequences reveal a special significance for nucleosome positioning of a motif consisting of approximately 10 bp periodic placement of TA dinucleotide steps. Contributions to histone binding and nucleosome formation from periodic TA steps are more significant than those from other periodic steps such as AA (=TT), CC (=GG) and more important than those from the other YR steps (CA (=TG) and CG), which are reported to have greater conformational flexibility in protein-DNA complexes even than TA. We report the development of improved procedures for measuring the free energies of even stronger positioning sequences that may be isolated in the future, and show that when the favorable free energy of histone-DNA interactions becomes sufficiently large, measurements based on the widely used exchange method become unreliable.

Animals↗
PubMed1998

TGGA repeats impair nucleosome formation.

Nucleosomes, the building blocks of chromatin, are responsible for DNA packaging in eukaryotic cell nuclei. They play a structural role in genome condensation, and influence transcription and replication. Properties of the DNA sequence, such as curvature and flexibility, direct the location of nucleosomes. DNA sequences that position nucleosomes have been identified and rules that govern their properties have been formulated. However, DNA sequences that are refractory to nucleosome formation have been less well characterised and it is possible that they may perturb or alter chromatin structure. Here we identify such sequences by selecting those that refrain from nucleosome formation from a large pool of synthetic DNA fragments with a central region of 146 random base-pairs fitted with adapters for PCR amplification. These were used for in vitro salt-induced reconstitution of nucleosomes under thermodynamic equilibrium conditions. Fragments that did not form nucleosomes were purified, amplified by PCR, and the reconstitution was repeated. After 17 rounds of negative selection, the material was highly enriched in sequences reluctant to form nucleosomes. Cloning and sequencing revealed that 35% of the molecules had long repeats of TGGA, and their affinity for histone octamers was about half that of average DNA.

Animals↗
PubMed1998

DNA tetraplex formation in the control region of c-myc.

The c-myc oncogene is one of the most commonly malfunctioning genes in human cancers, and is an attractive target for anti-gene therapy. Although synthetic oligonucleotides designed to silence c-myc expression via one of its major control elements function well in vitro, their mode of action has been indefinite. Here we show that the targeted control element adopts an intrastrand fold-back DNA tetraplex, which requires potassium ions for stability in vitro. We believe formation of the tetraplex is important for c-myc activation in vivo, and propose a transcription initiation mechanism that explains how anti-gene therapy silence c-myc at the molecular level.

Base Sequence↗
PubMed1998

The interactions between the fluorescent dye thiazole orange and DNA.

The interaction of the fluorescent dye thiazole orange (TO) with nucleic acids is characterized. It is found that TO binds with highest affinity to double-stranded (ds) DNA [log (K) approximately 5.5 at 100 mM salt], about 5-10 times weaker to single-stranded polypurines, and further 10-1000 times weaker to single-stranded polypyrimidines. TO binds as a monomer to dsDNAs and poly(dA), both as a monomer and as a dimer to poly(dG) and mainly as a dimer to poly(dC) and poly(dT). The fluorescence quantum yield of TO free in solution is about 2 x 10(-4), and it increases to about 0.1 when bound to dsDNA or to poly(dA), and to about 0.4 when bound to poly(dG). Estimated quantum yields of TO bound to poly(dC) and poly(dT) are about 0.06 and 0.01, respectively. The quantum yield of bound TO depends on temperature and decreases about threefold between 5 and 50 degrees C.

Benzothiazoles↗
PubMed1998

Characterization of fluorescein-oligonucleotide conjugates and measurement of local electrostatic potential.

The properties of fluorescein are substantially altered upon conjugation to nucleic acids, affecting not only the molar absorptivities and fluorescence quantum yields but also the protolytic equilibrium constant and fluorescence lifetimes. Around neutral pH, the fluorescein moiety is present as both mono- and dianion, and the pKa relating them is increased from 6.43 for free fluorescein to about 6.90 for fluorescein attached to both single- and double-stranded oligonucleotides of at least 12 bases/base pairs. This difference reflects the local electrostatic potential around the nucleic acid, which is calculated to -28 mV. The molar absorptivities and spectral responses of the conjugated fluorescein protolytic species are also determined, from which the concentrations of fluorescein-oligonucleotide conjugates can be calculated by assuming: epsilon 494 = 62000/[1 + 10-(pH-6.90)] + 12000/[1 + 10(pH-6.90)] (M-1 cm-1). The fluorescence quantum yield of the conjugates depends, in a complex way, on temperature, environment and oligonucleotide length, sequence and conformation, and must be determined for each experimental situation.

Fluoresceins↗
PubMed1997

Identification and characterization of genomic nucleosome-positioning sequences.

Positioned nucleosomes are believed to play important roles in transcriptional regulation and for the organization of chromatin in cell nuclei. Here, we have isolated the DNA segments in the mouse genome that form the most stable nucleosomes yet characterized. In separate molecules we find phased runs of three to four adenine nucleotides, extensive CA repeats, and in a few cases phased TATA tetranucleotides. The latter forms the most stable nucleosome yet characterized. One sequence with CAG repeats was also found. By fluorescence in situ hydridization the selected sequences are shown to be localized at the centromeric regions of mouse metaphase chromosomes.

Animals↗
PubMed1994

Absorption flattening in the optical spectra of liposome-entrapped substances.

Measuring optical spectra of chromophore molecules entrapped in liposomes can lead to considerable distortion because of absorption flattening. This phenomenon is analyzed theoretically, and it is shown that the deviation from the Beer-Lambert law becomes larger as the molar absorptivity of the chromophore increases and as the number of entrapped molecules becomes larger. The theoretical equations are tested experimentally with intermediate-sized phospholipid vesicles containing entrapped cytochrome c. It is shown that considerable absorption flattening is observed with vesicles containing about 50 chromophore molecules. The equation given can be used to correct the spectrum.

Absorption↗