The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation

Horizontal gene transfer shapes the genomes of prokaryotes by allowing rapid acquisition of novel adaptive functions. Conjugation allows the broadest range and the highest gene transfer input per transfer event. While conjugative plasmids have been studied for decades, the number and diversity of integrative conjugative elements (ICE) in prokaryotes remained unknown. We defined a large set of protein profiles of the conjugation machinery to scan over 1,000 genomes of prokaryotes. We found 682 putative conjugative systems among all major phylogenetic clades and showed that ICEs are the most abundant conjugative elements in prokaryotes. Nearly half of the genomes contain a type IV secretion system (T4SS), with larger genomes encoding more conjugative systems. Surprisingly, almost half of the chromosomal T4SS lack co-localized relaxases and, consequently, might be devoted to protein transport instead of conjugation. This class of elements is preponderant among small genomes, is less commonly associated with integrases, and is rarer in plasmids. ICEs and conjugative plasmids in proteobacteria have different preferences for each type of T4SS, but all types exist in both chromosomes and plasmids. Mobilizable elements outnumber self-conjugative elements in both ICEs and plasmids, which suggests an extensive use of T4SS in trans. Our evolutionary analysis indicates that switch of plasmids to and from ICEs were frequent and that extant elements began to differentiate only relatively recently. According to the present results, ICEs are the most abundant conjugative elements in practically all prokaryotic clades and might be far more frequently domesticated into non-conjugative protein transport systems than previously thought. While conjugative plasmids and ICEs have different means of genomic stabilization, their mechanisms of mobility by conjugation show strikingly conserved patterns, arguing for a unitary view of conjugation in shaping the genomes of prokaryotes by horizontal gene transfer

The anti-apoptotic effect of leukotriene D-4 involves the prevention of caspase 8 activation and Bid cleavage

We have shown in a previous study that leukotriene D-4 (LTD4) signalling increases cell survival and proliferation in intestinal epithelial cells [ohd, Wikstrom and Sjolander (2000) Gastroenterology 119, 1007-1018]. This is highly interesting since inflammatory conditions of the bowel are associated with an increased risk of developing colon cancer. The enzyme cyclooxygenase 2 (COX-2) is important in this context since it is upregulated in colon cancer tissues and in tumour cell lines. Treatment with the COX-2-specific inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methane sulphonamide has been shown previously to cause apoptosis in intestinal epithelial cells. In the present study, we attempted to elucidate the underlying mechanisms and we can now show that a mitochondrial pathway is employed. Inhibition of COX-2 causes release of cytochrome c, as shown by both Western-blot and microscopy studies, and as with apoptosis, this is significantly decreased by LTD4. Since previous studies showed increased Bcl-2 levels on LTD4 stimulation, we further studied apoptotic regulation at the mitochondrial level. From this we could exclude the involvement of the anti-apoptotic protein Bcl-X-L as well as its pro-apoptotic counterpart Bax, since they are not expressed. Furthermore, the activity of the proapoptotic protein Bad (Bcl-2/Bcl-X-L-antagonist, causing cell death) was completely unaffected. However, inhibition of COX-2 caused cleavage of caspase 8 into a 41 kDa fragment associated with activation and caused the appearance of an activated 15 kDa fragment of Bid. This indicates that N-(2-cyclohexyloxy4-nitrophenyl)methane sulphonamide-induced apoptosis is mediated by the activation of caspase 8, via generation of truncated Bid, and thereafter release of cytochrome c. Interestingly, LTD4 not only reverses the effects induced by inhibition of COX-2 but also reduces the apoptotic potential by lowering the basal level of caspase 8 activation and truncated Bid generation

Endogenous production of leukotriene D(4) mediates autocrine survival and proliferation via CysLT(1) receptor signalling in intestinal epithelial cells.

The cysteinyl leukotriene(1) (CysLT(1)) receptor (CysLT(1)R) enhances survival and proliferation of intestinal cells via distinct pathways. Here, we have demonstrated that there is significant endogenous production of CysLTs from both non-tumour-and tumour-derived intestinal epithelial cells. Treatment of two non-tumour cell lines, Int 407 and IEC-6, with CysLT1R antagonists led to shrinkage and detachment of cells, confirmed as apoptotic cell death, and a dose-dependent reduction in proliferation. However, in the tumour intestinal cell lines Caco-2, SW480, HCT-116 and HT-29, treatment with CysLT1R antagonists significantly reduced proliferation, but had no effect on apoptosis. A unique characteristic of intestinal cancer cells is the presence of nuclear CysLT(1)Rs, which are inaccessible to receptor antagonists. In these cells, inhibition of the endogenous production of CysLTs indirectly, by 5-lipoxygenase inhibition, impaired CysLT1R signalling throughout the cell, and resulted in apoptosis of the tumour cells. These data reveal the existence of constitutive CysLT1R signalling that mediates both survival and proliferation in intestinal cells. Importantly, we propose that tumour-derived intestinal cells are resistant to CysLT(1)R antagonist-induced apoptosis, a phenomena that could be explained by nuclear CysLT1R signalling

Evaluation of the Effect of Average Annual Temperatures in Slovakia between 1971 and 2020 on Stresses in Rigid Pavements

The scientific community in Central Europe often discusses the extent to which temperature changes over the last two decades have contributed to changing the stresses induced in structures. In the field of road and environmental engineering, this question is especially pertinent for pavements. The pavement structure must first be defined methodologically by identifying and defining the types of parameters that change with time. Additionally, it is important to identify the areas of Central Europe that are most affected by climate change. The most important parameters must be described statistically for these areas. Slovakia is one of the countries that may be able to contribute to the solution of this issue due to its location in the middle of Europe. This paper provides a statistical analysis for the period from 1971 to 2020 in Slovakia. A concrete pavement, which is the most commonly used type of pavement, must be used as an example to numerical assess the situation. The conclusions and discussion in this scientific field are directed towards the evaluation of the measurement results in the context of the designed pavement composition and the calculations using the different methods specified in the standards

The effect of propofol on actin, ERK-1/2 and GABA(A) receptor content in neurones

Aim: Interaction with the gamma-aminobutyric acid receptor (GABA(A)R) complex is recognized as an important component of the mechanism of many anaesthetic agents, including propofol. The aims of this study were to investigate the effect of propofol on GABA(A)R, to determine whether exposure of neurones to propofol influences the localization of GABA(A)R within the cell and to look for cytoskeletal changes that may be connected with activation, such as the mitogen-activated protein kinase (MAPK) pathway. Methods: Primary cortical cell cultures from rat, with and without pre-incubation with the GABA(A)R antagonist bicuculline, were exposed to propofol. The cells were lysed and separated into membrane and cytosolic fractions. Immunoblot analyses of filamentous actin (F-actin), the GABA(A) beta(2)-subunit receptor and extracellular signal-regulated kinase-1/2 (ERK-1/2) were performed. Results: Propofol triggers an increase in GABA(A)R, actin content and ERK-1/2 phosphorylation in the cytosolic fraction. In the membrane fraction, there is a decrease in GABA(A) beta(2)-subunit content and an increase in both actin content and ERK-1/2 phosphorylation. The GABA(A)R antagonist bicuculline blocks the propofol-induced changes in F-actin, ERK and GABA(A) beta(2)-subunit content, and ERK-1/2 phosphorylation. Conclusion: We believe that propofol triggers a dose-dependent internalization of the GABA(A) beta(2)-subunit. The increase in internal GABA(A) beta(2)-subunit content exhibits a close relationship to actin polymerization and to an increase in ERK-1/2 activation. Actin contributes to the internalization sequestering of the GABA(A) beta(2)-subunit

Crosstalk between colon cancer cells and macrophages via inflammatory mediators and CD47 promotes tumour cell migration.

Tumour-associated macrophages (TAMs) of the M2 phenotype are present in the stroma of many tumours and are frequently associated with the progression of several types of cancer. We investigated the role of M2 macrophages in colon cancer progression and found that human colon cancer tissue had elevated numbers of CD68(+) (macrophage marker) cells and CD206(+) (M2 macrophage marker) cells and increased CD47 expression. To explore potential interplay between colon cancer cells and M2 macrophages, we differentiated the monocyte cell line THP-1 into M1 and M2 macrophages (CD206(high) and Th2 cytokine-secreting cells), respectively. M2 macrophages migrated faster than M1 macrophages towards SW480-conditioned medium. Similarly, M2 macrophage-conditioned medium induced SW480 cell migration and CD47 expression. Factors released by macrophages were involved in this induction. In addition, SW480 cells migrated faster when co-cultured with M2 macrophages. Inhibition of CD47 with blocking antibodies or siRNA significantly reduced the migration of SW480 cells in the presence of M2 macrophages. This effect was further decreased via blocking antibodies against the CD47 ligand signal-regulatory protein α (SIRPα). Additionally, cancer cells also secreted significant levels of IL-10, thereby promoting M2 macrophage differentiation. These findings indicate that a TAM-enriched tumour microenvironment promotes colon cancer cell migration and metastasis

Evaluation of the Effect of Average Annual Temperatures in Slovakia between 1971 and 2020 on Stresses in Rigid Pavements

The scientific community in Central Europe often discusses the extent to which temperature changes over the last two decades have contributed to changing the stresses induced in structures. In the field of road and environmental engineering, this question is especially pertinent for pavements. The pavement structure must first be defined methodologically by identifying and defining the types of parameters that change with time. Additionally, it is important to identify the areas of Central Europe that are most affected by climate change. The most important parameters must be described statistically for these areas. Slovakia is one of the countries that may be able to contribute to the solution of this issue due to its location in the middle of Europe. This paper provides a statistical analysis for the period from 1971 to 2020 in Slovakia. A concrete pavement, which is the most commonly used type of pavement, must be used as an example to numerical assess the situation. The conclusions and discussion in this scientific field are directed towards the evaluation of the measurement results in the context of the designed pavement composition and the calculations using the different methods specified in the standards

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D(4) in intestinal epithelial cells.

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT(1)R and CysLT(2)R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-alpha), and leukotriene D(4) (LTD(4)), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC(4) synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-alpha and LTD(4), to a lesser extent, up-regulate the CysLT(1)R levels. Interestingly, TNF-alpha also reduced CysLT(2)R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression

Leukotriene D-4 activates MAPK through a Ras-independent but PKC epsilon-dependent pathway in intestinal epithelial cells

We have recently shown that leukotriene D-4 (LTD4) increases cell survival in intestinal epithelial cells. Here we report and explore the complementary finding that LTD4 also enhances proliferation in these cells. This proliferative response was approximately half of that induced by epidermal growth factor (EGF) and its required activation of protein kinase C (PKC), Ras and the mitogen-activated protein kinase (MAPK) Erk-1/2. EGF also activated Erk-1/2 in these cells; however the EGF-receptor inhibitor PD153035 did not affect the LTD4-induced activation of Erk-1/2. In addition, LTD4 did not induce phosphorylation of the EGF receptor, nor did pertussis toxin (PTX) block EGF-induced activation of Erk-1/2, thus refuting a possible crosstalk between the receptors. Furthermore, LTD4-induced, but not EGF-Induced, activation of Erk-1/2 was sensitive to PTX, PKC inhibitors and downregulation of PKCepsilon. A definite role for PKCepsilon in LTD4-induced stimulation of Erk-1/2 was documented by the inability of LTD4 to activate Erk-1/2 in cells transfected with either the regulatory domain of PKCepsilon (an isoform specific dominant-negative inhibitor) or a kinase-dead PKCepsilon Although Ras and Raf-1 were both transiently activated by LTD4, only Raf-1 activation was abolished by abrogation of the PKC signal. Furthermore, the LTD4-induced activation of Erk-1/2 was unaffected by transfection with dominant-negative N17 Ras but blocked by transfection with kinase-dead Raf-1. Consequently, LTD4 regulates the proliferative response by a distinct Ras-independent, PKCepsilon-dependent activation of Erk-1/2 and a parallel Ras-dependent signaling pathway