Hybrid genes in the analysis of transformation conditions: II. Transient expression vs stable transformation—analysis of parameters influencing gene expression levels and transformation efficiency

Reports on direct gene transfer have dealt with either the obtention of stable transformants and transgenic plants, or described the use of reporter genes to analyse different aspects of gene expression in plant protoplasts and conditions for their use in transient gene expression assays.
In this paper we present comparisons between several transformation techniques, show species-specific differences in efficiencies of stable transformants and in the levels of transient gene expression, and report on the identification of major parameters responsible for DNA uptake as judged from transient chloramphenicol acetyl transferase (CAT) expression levels and from efficiencies of transformation based on kanamycin-resistance. The described procedures have been simplified, optimized and standardized and should allow routine use with a great variety of plant species.     

Behavioural and physiological mechanisms behind extreme longevity in Daphnia

The combined effect of external environment and energy allocation strategy of the organism on longevity can be exceptional. In a cold oligotrophic fishless habitat, individual Daphnia can live for over a year, several times the usual Daphnia lifespan. This extreme lifespan is in part a consequence of the overwintering strategy which includes storing resources and delaying reproduction until another spring. Yet, contrasting strategies may be applied by Daphnia, resulting in over twofold differences in lifespan within a single habitat. We identify physiological mechanisms mediating such differences in longevity in closely related Daphnia of two lineages coexisting within a high altitude lake, testing the predictions that long-lived animals stay in colder waters and have lower metabolic rates, irrespective of temperature. Vertical distribution of the animals was assessed during three summer stratification seasons, and metabolic activity was measured as oxygen consumption and RNA:DNA ratio. The results not only support our predictions but also reveal that habitat choice is dependent on reproductive status rather than genotype. The young individuals of the overwintering lineage may delay reproduction in part by staying in colder waters than the reproducing adults, which together with low intrinsic metabolic rates may underlie the longevity of Daphnia of this lineage.     

Complex of Rutin with β-Cyclodextrin as Potential Delivery System

This study aimed to obtain and characterize an RU-β-CD complex in the context of investigating the possibility of changes in the solubility, stability, antioxidative and microbiological activity as well as permeability of complexated rutin as against its free form. The formation of the RU-β-CD complex via a co-grinding technique was confirmed by using DSC, SEM, FT-IR and Raman spectroscopy, and its geometry was assessed through molecular modeling. It was found that the stability and solubility of the so-obtained complex were greater compared to the free form; however, a slight decrease was observed inits antibacterial potency. An examination of changes in the EPR spectra of thecomplex excluded any reducing effect of complexation on the antioxidative activity of rutin. Considering the prospect of preformulation studies involving RU-β-CD complexes, of significance is also the observed possibility of prolongedly releasing rutin from the complex at a constant level over along period of 20 h, and the fact that twice as much complexated rutin was able topermeate compared to its free form.     

Genetic factors predisposing to the development of papillary thyroid cancer

According to classic theory of neogenesis, cancer arises from well-differentiated cell that in response to variety of factors de-differentiates, becomes able to proliferate without control and/or loses its ability to undergo apoptosis. According to another theory, cancers (at least cancers of some organs) originate from stem cells, which "by definition" are poorly differentiated and able to proliferate indefinitely. Therefore a lower number of abnormal events is necessary for these cells to escape proliferation-controlling mechanisms. With regard to papillary thyroid cancers it is still thought that it arises from well-differentiated thyreocyte. One of the characteristic features of cancer cell is chromosomal instability. Lowest number of such abnormalities is observed in well-differentiated thyroid cancers (including papillary cancer), intermediate - in poorly-differentiated cancers, while highest - in anaplastic cancers. Microarray analysis shows that despite of clinical heterogeneity, gene expression profiles of papillary cancers are very similar. Genetic anomalies predisposing to the development of papillary cancer most commonly regard proteins that possess kinase activity. Kinases phosphorylate other proteins, and play an extremely important role in signal transduction from outside the cell as well as inside the cell. Constitutive activation of some kinases may lead to the excessive and/or permanent activation of some transduction pathways specific for mitogens or growth factors. This results in excessive proliferation. The best known protein of such type which function is altered in papillary thyroid cancers is RET - a membrane-located growth factor-receptor with kinase activity. RET gene undergoes different rearrangements in this type of cancer. There are approximately 10 RET rearrangements known, with RET/PTC3 and RET/PTC1 being most common. In this anomaly kinase domain-encoding 3' end of RET gene is aberrantly bound to 5' end of another gene. Fusion protein synthesized on such hybrid template is not present in the cell membrane but in the cytoplasm, where it permanently activates transduction pathway specific for RET. NTRK1 gene encoding a member of family of neuronal growth factor receptors containing thyrosine kinase domain is also rearranged in papillary cancers. However, genes fused to its kinase domain-encoding sequence are different from the ones fused to RET. MET, a gene encoding another membrane protein with thyrosine kinase activity, which acts as a growth factor-receptor, is overexpressed in 70%-90% of papillary thyroid cancers. BRAF gene encoding another yet kinase transducing signals from RAS and RAF to the cell is mutated at position 1796 (T/A, amino acid substitution V599E) in 38-69% of papillary cancers. The presence of this activatory mutation is associated with higher degree of clinical advancement of the disease. In addition, in majority of papillary cancers tested, mutations of the genes encoding nuclear triiodothyronine receptors were found. Transgenic mice with both TRB allele replaced with dominant-negative TRB mutants develop aggressive thyroid cancers. Progression from papillary to anaplastic cancer is most possibly caused by the occurrence of additional anomalies within P53, RAS, NM23,b-catenin gene and other genes.     

Characterization of vaginal lactobacilli in women after kidney transplantation

Limited number of publications described vaginal microflora after kidney transplantation. Our PubMed search revealed only 18 publications including words “vaginal bacteria &; kidney transplant” in the period of 1978–2011. The aim of this study was to characterize lactobacilli isolated from vaginal swabs of women after kidney transplantation, compared with healthy women. Eighteen renal transplant recipients (mean age 36.1) and 20 healthy women (mean age 36.0) were evaluated. Lactobacilli were cultured on MRS and Columbia blood agars. Biochemical identification with API 50 CHL (bioMerieux, Marcy L’Etoile, France) and multiplex PCR according to Song et al. was performed. Lactobacilli were tested for production of H₂O₂. Minimal inhibitory concentrations (MICs) of selected antimicrobial agents were determined with E-tests (bioMerieux, Marcy L’Etoile, France) and interpreted with CLSI and EUCAST criteria. No bacterial vaginosis was found among studied women. Two strains of group I were identified as Lactobacillus delbrueckii; 18 strains as Lactobacillus gasseri and 15 strains as Lactobacillus crispatus. Only 3 strains from group II were not identified by species-specific mPCR. Group IV was represented with 2 unidentified strains. Vaginal lactobacilli isolated from healthy women represented more homogenous group compared with heterogenous renal transplant recipients. Biochemical identification of lactobacilli by API 50 CHL kits was concordant with mPCR results only in 7 cases (17.5%), all 7 strains were identified as L. crispatus. Majority (93%) of lactobacilli were H₂O₂ producers. All isolated lactobacilli (100%) demonstrated high resistance to metronidazole (MIC > 256 μg/ml). Only 2 strains resistant to vancomycin (MICs: 32 and 256 μg/ml respectively), in the study and control group, and one to moxifloxacin (MIC = 32 μg/ml), were found. Resistance to metronidazole and vancomycin was concordant in CLSI and EUCAST (2010) criteria. Although significant differences between lactobacilli isolated from vaginas of kidney transplant and healthy women were not demonstrated, we demonstrated strains resistant to metronidazole, vancomycin and moxifloxacin in groups of examined women. Our study was performed on a small group of kidney transplant recipients and further more detailed molecular studies on a larger group of patients are required to confirm our results.     

Genes and structure of selected cytokines involved in pathogenesis of psoriasis

Psoriasis is a common skin disease involving 1-4% of human population worldwide, of strong genetic background. The following cytokines are directly involved in psoriasis: TNF, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-18, IL-19, IL-20, IL-23 whereas IL-4, IL-10, IL-12 as well as IL-11, IL-17 and IFN-gamma are rather indirectly engaged. This work is a review of some genetic factors and structure of selected cytokines and receptors and their genes location.     

Computational Effective Fault Detection by Means of Signature Functions

The paper presents a computationally effective method for fault detection. A system’s responses are measured under healthy and ill conditions. These signals are used to calculate so-called signature functions that create a signal space. The current system’s response is projected into this space. The signal location in this space easily allows to determine the fault. No classifier such as a neural network, hidden Markov models, etc. is required. The advantage of this proposed method is its efficiency, as computing projections amount to calculating dot products. Therefore, this method is suitable for real-time embedded systems due to its simplicity and undemanding processing capabilities which permit the use of low-cost hardware and allow rapid implementation. The approach performs well for systems that can be considered linear and stationary. The communication presents an application, whereby an industrial process of moulding is supervised. The machine is composed of forms (dies) whose alignment must be precisely set and maintained during the work. Typically, the process is stopped periodically to manually control the alignment. The applied algorithm allows on-line monitoring of the device by analysing the acceleration signal from a sensor mounted on a die. This enables to detect failures at an early stage thus prolonging the machine’s life.