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In this paper a creation of new numerical set-theoretical constructions of Boolean functions with their properties has been presented. The parted conjuncterms and decomposition clones which can be used for realization of different optimization logic synthesis of digital devices and systems have been proposed. Set theoretical operations and procedures with these constructions have been considered. Examples which illustrate simplicity of their practical realization have been presented. Moreover, the paper includes the comparison of new results obtained on the basis of the proposed approach with the results obtained by means of commercial software Quartus II of Altera.

Background: The Pl A2 allele of the gene encoding for GPIIIa subunit of the platelet membrane receptor glycoprotein (GP) IIb/IIIa has been suggested as a significant risk factor for thrombotic complications of coronary artery disease (CAD). The aim of the current investigation was to investigate the association between Pl A GPIIIa polymorphism and the extent of angiographically confirmed CAD in patients from the north region of Poland. Methods: The study was performed in 397 male Caucasian patients. All subjects had significant coronary artery stenosis confirmed by elective coronary angiography. Screening for the Pl A GPIIIa genotypes was performed by polymerase chain reaction of genomic DNA, followed by Nci I digestion and agarose gel electrophoresis. Results: The genotype distribution of the Pl A GPIIIa polymorphism in our study group was Pl A1/A1 —75%, Pl A1/A2 —24% and Pl A2/A2 —1% with Pl A1 and Pl A2 allele frequencies of 0.87 and 0.13, respectively. The prevalence of the homozygous Pl A1/A1 genotype among subjects with multiple-vessel CAD (two or three vessels with at least 50% stenosis) was significantly higher than in patients with single-vessel disease; the odds ratio of Pl A2/A2 or Pl A1/A2 patients for having multiple-vessel CAD was 0.46 (95% CI 0.27–0.77, P Pl A1/A1 patients was significantly higher in comparison to Pl A2/A2 and Pl A1/A2 patients (7.58±2.20 and 6.98±2.37, respectively, P Conclusions: Our results suggest, that the Pl A1/A1 genotype of Pl A GPIIIa polymorphism is associated with more severe CAD in male Caucasian patients from the north region of Poland.

Most eukaryotic cell types can withdraw from proliferative cell cycles and remain quiescent for extended periods. Intact nuclei isolated from quiescent murine NIH3T3 cells fail to replicate in vitro when incubated in Xenopus egg extracts, although intact nuclei from proliferating cells replicate well. Permeabilization of the nuclear envelope rescues the ability of quiescent nuclei to replicate in the extract. We show that origin replication complex (ORC), minichromosome maintenance (MCM), and Cdc6 proteins are all present in early quiescent cells. Immunodepletion of Cdc6 or the MCM complex from Xenopus egg extract inhibits replication of permeable, quiescent, but not proliferating, NIH3T3 nuclei. Immunoblotting results demonstrate that mouse homologues of Mcm2, Mcm5, and Cdc6 are displaced from chromatin in quiescent cells. However, this absence of chromatin-bound Cdc6 and MCM proteins from quiescent cells appears not to be due to the absence of ORC subunits as murine homologues of Orc1 and Orc2 remain chromatin-bound in quiescent cells. Surprisingly, intact quiescent nuclei fail to bind exogenously added XCdc6 or to replicate in Xenopus egg extracts immunodepleted of ORC, even though G1- or S-phase nuclei still replicate in these extracts. Our results identify Cdc6 and the MCM complex as essential replication components absent from quiescent chromatin due to nonfunctional chromatin-bound ORC proteins. These results can explain why quiescent mammalian nuclei are unable to replicate in vivo and in Xenopus egg extracts.

We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.

Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.

The origin recognition complex (ORC) is required to initiate eukaryotic DNA replication and also engages in transcriptional silencing in S. cerevisiae. We observed a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalized with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 were found in physical complex. The association was shown biochemically to require the chromodomain and shadow domains of HP1. The amino terminus of DmORC1 contained a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Finally, heterozygous DmORC2 recessive lethal mutations resulted in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors.

Activating mutations in the Ki-ras2 oncogene are frequently observed in sporadic colorectal adenomas and their incidence is reported to rise in large and tubulovillous adenomas to values close to those in carcinomas. This study shows that this property is a feature of adenomas growing in large bowel that has already demonstrated its propensity to engender malignant tumours: i.e., bowel in which there is a synchronous carcinoma. Adenomas from cancer-free bowel do not share this high incidence of Ki-ras mutations. This difference in mutation incidence between adenomas from cancer-free and cancer-bearing patients does not appear to derive from sampling bias relative to adenoma size, site, or patient age, nor is it found in another gene (APC) known to be of importance in adenoma formation. Large, dysplastic adenomas from cancer-bearing bowel, however, are particularly liable to carry Ki-ras mutations when they arise in patients over 70 years old. The observations suggest that the role of Ki-ras mutations may be more subtle than merely enhancing adenoma growth. Adenoma cells of cancer-prone individuals may suffer more mutational events than those in persons selected as cancer-free.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.

Carbon monoxide (CO) intoxications still remain an important clinical problem. Under an influence of CO the cellular respiration is inhibited. Structures of the central nervous system (CNS), extremely vulnerable to hypoxia, are usually considerably damaged. In result, a variety of pathologic neurological symptoms may appear and characteristic biphasic course of complaints is often observed. The modern methods of neuroimaging (CT, MRI, SPECT, PET) enable to show a connection between found structural injury and clinical symptoms, and when repeatedly performed allow us to evaluate the dynamics of the CNS dysfunction. Because of late complications, which may occur after the period of latency, the authors stress the value of the prolonged (for up to few years) ambulatory observation of subjects after CO intoxication.

The objective of the present study was to analyse the potential synergistic influence of the insertion/deletion polymorphism of the angiotensin-converting enzyme gene (I/D ACE) and the A1166C polymorphism of the angiotensin-II type 1 receptor gene polymorphisms (A1166C AT1R) on the left ventricular size and performance. Three hundred sixty and one consecutive, Caucasian patients with angiographically confirmed coronary artery disease (CAD) were enrolled into the study. Left ventricular diameter, mass and function were evaluated by echocardiography. Screening for the I/D ACE and A1166C AT1R genotypes was performed by polymerase chain reaction of genomic DNA, followed by restriction enzyme digestion and agarose gel electrophoresis. The I/D ACE and A1166C AT1R genotypes separately were not significantly associated with the left ventricular size and function parameters in CAD patients. However, trends towards decreased left ventricular ejection fraction (LVEF) as well as increased left ventricular end-diastolic diameter (LVEDD) and left ventricular mass index (LVMI) were observed when patients with genotype DD+CC/AC and DD+CC were compared to patients homozygous only in one locus (DD or CC). Significant increase in LVEDD and LVMI was observed only in patients with a history of anterior myocardial infarction with combined genotype DD+CC/AC or DD+CC. This study does not support the role of the ACE I/D and AT1R A1166C polymorphisms in the determination of the left ventricular size and performance in patients with significant coronary atherosclerosis. However, it indicates that the influence of polymorphisms may be present in specific patient populations.

Background There is growing evidence from recent studies that atrial natriuretic peptide (ANP) plays an important part in coronary blood flow regulation and in atherosclerosis. Transition T2238→C in the atrial natriuretic peptide ( ANP ) precursor gene, which leads potentially to the translation of ANP with 2 additional arginines, has been suggested to be associated with salt-sensitive hypertension. According to our knowledge, this study is the first to look for the potential association of the ScaI ANP gene polymorphism with the history of nonfatal myocardial infarction and the extent of coronary artery disease (CAD). Methods The study was performed in 847 consecutive, white patients (719 men and 128 women) with significant coronary artery stenosis confirmed by means of elective coronary angiography (at least 1 coronary artery with ≥50% lumen narrowing). Screening for the T2238→C substitution was performed by means of polymerase chain reaction of genomic DNA, followed by ScaI digestion and agarose gel electrophoresis. Results We found a significant association of the A2A2 ScaI ANP genotype with a higher incidence of positive history of nonfatal myocardial infarction (odds ratio 1.85, 95% CI 1.33-2.58) and multiple-vessel CAD (odds ratio 1.45, 95% CI 1.02-2.06). The ScaI ANP genotype distribution did not differ with age, sex, body mass index, plasma lipids, hypertension, diabetes mellitus, and family history of CAD in studied groups. Conclusions Our results suggest that the ScaI ANP polymorphism may be associated with nonfatal myocardial infarction and the extent of CAD. However, the precise mechanism of this association remains to be determined. (Am Heart J 2003;145:125-31.)

Cdc6 is essential for eukaryotic DNA replication. We have mutated highly conserved CDK phosphorylation sites in Cdc6. Contrary to their reported phenotypes in human cells, unphosphorylatable ΔCDK mutants fully support DNA replication in Xenopus eggs. WtCdc6 is actively exported from the nucleus, which could explain why nuclear permeabilization is required for reinitiation within one cell cycle. However, ΔCDK mutants are retained in the nucleus, yet surprisingly they still support only one round of replication. As these highly conserved CDK sites are unnecessary for replication once per cell cycle, an alternative checkpoint role for monitoring completion of the S phase is suggested.

The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G(1)/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21(Cip1). This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2 x cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.

Minichromosome maintenance (Mcm) proteins and the constituents of the origin recognition complex (Orc) are essential components of the eukaryotic replication initiation apparatus. Published evidence strongly suggests that the binding of Mcm proteins to chromatin is contingent upon the prior binding of Orc proteins. Here we use two different approaches to investigate the presence of the human ORC2 protein and of Mcm proteins on chromatin of HeLa cells in various cell cycle phases. First, we mobilized chromatin-bound proteins by micrococcal nuclease and analyzed the resulting digestion products by sucrose gradient centrifugations. Under digestion conditions when Mcm proteins were almost entirely released from chromatin, ORC2 protein was found to be associated with chromatin fragments containing several hundred base pairs of DNA. Second, we used an in vivo cross-linking procedure to covalently link Mcm proteins and ORC2 to DNA by short exposure of intact HeLa cells to formaldehyde. Specific immunoprecipitations revealed that cross-linked nucleoprotein fragments carried either Mcm proteins or ORC2 protein, but not both. Based on the lengths of the DNA fragments in immunoprecipitates, we estimate that the distance between chromatin-bound ORC2 protein and chromatin-bound Mcm proteins must be at least 500-1000 base pairs in HeLa cells.

Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. We show here that H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopussperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development.

An important aspect of cell behaviour is that DNA replication happens only once per cell cycle. Replicated DNA is unable to re-replicate until cell division has occurred. Unreplicated DNA is in a replication-competent or 'licensed' state. The ability to replicate is lost in S phase and regained following passage through mitosis. Recent evidence has implicated an MCM (minichromosome maintenance) protein complex and the Cdc6 protein in determining replication competence. Regeneration of replication competence upon passage through mitosis entails changes in protein kinase activity, of which the MCMs are a likely target. Features of the mechanism that restricts DNA replication to once per cell cycle appear to be conserved throughout eukaryotes.

DNA replication in the budding yeast Saccharomyces cerevisiae initiates from origins of specific DNA sequences during S phase. A screen based on two- and one-hybrid approaches demonstrates that the product of the DBF4 gene interacts with yeast replication origins in vivo. The Dbf4 protein interacts with and positively regulates the activity of the Cdc7 protein kinase, which is required for entry into S phase in the yeast mitotic cell cycle. The analysis described here suggests a model in which one function of Dbf4 may be to recruit the Cdc7 protein kinase to initiation complexes.

Two age groups--young (19-35) and elderly (70-91)--were compared with respect to natural killer (NK) cytotoxic activity. In both groups, low and high NK responders could be distinguished. Low NK responders constituted about 70% of all elderly and 40% of young individuals. The differences in the magnitude of NK activity among the young and elderly groups could only be observed when peripheral-blood mononuclear cells but not peripheral-blood lymphocytes were used as effector cells in a 51Cr release assay. Experiments with removal or addition of graded numbers of monocytes showed that these cells were responsible for the low level of NK activity in both the young and elderly low NK responders.

The present paper discusses the importance of needs analysis in ESP. By delving into different theories, the author’s overall objective is to scrutinize a wide spectrum of existing frameworks that would lead to a single, comprehensive model for needs assessment. The impact of the said approaches on course design is underlined with the particular example of Business English. Ultimately, the author unveils a framework which could serve as a basis for syllabus design in all types of ESP courses.

The aim of the present paper is to analyse the extent to which contemporary e-learning solu-tions support the development of intercultural competence in the didactics of specialist lan-guages. A special consideration will be given to those solutions which use e-learning modules. A few remarks concerning the terminology of intercultural competence as well as didactic modules have been made at the beginning. Last but not least, on the basis of the theory of didactic module, the process of (glotto) didactic module development and the examples of its realisation in the context of development of intercultural competence have been indicated. As shown in the paper e-learning solutions based on glottodidactic modules contribute to the de-velopment of intercultural competence, which is an essential component equipping interactor with indispensable and critical knowledge and skills. All this is required in modern specialised communication, occurring especially in multilingual and/or multicultural environments.

Background The origin recognition complex (ORC) and the minichromosome maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential for DNA replication. Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus . The Xenopus MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essential for DNA replication. Here, we describe the different but interdependent functions of the ORC and MCM complexes in DNA replication in Xenopus egg extracts. Results The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts. Immunodepletion of ORC inhibits DNA replication of Xenopus sperm nuclei. Mixing MCM-depleted and ORC-depleted extracts restores replication capacity. ORC does not co-localize with sites of DNA replication during elongation. However, at initiation the two staining patterns overlap. In contrast to MCMs, which are displaced from chromatin during S phase, XOrc1 and XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis. Permeable HeLa G1- and G2-phase nuclei can replicate in ORC-depleted extract, consistent with the presence of chromatin-bound ORC in both pre-replicative and post-replicative nuclei. Interestingly, the binding of ORC to chromatin does not require the presence of MCMs; however, the binding of MCM proteins to chromatin is dependent on the presence of ORC. Conclusions The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S. cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis. The binding of MCM proteins requires the presence of ORC. Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA.