Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy.     

The effects of nitric oxide synthase--versus lipoxygenase inhibition on coronary flow and nitrite outflow in isolated rat heart

The aim of this study was to assess the changes of coronary flow (CF) and nitrite outflow under inhibition of nitric oxide synthase (NOS) by Nomega-nitro-L-arginine monomethyl ester (L-NAME) or lipoxygenase (LOX) induced by nordihydroguaiaretic acid (NDGA) in isolated rat heart. The hearts of male Wistar albino rats (n=18, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff's technique at gradually increased constant coronary perfusion pressure (CPP) conditions (40-120 cm H2O) which induced flow-dependent nitric oxide (NO) release (nitrite outflow). The experiments were performed during control conditions, in the presence of NO synthesis inhibitor L-NAME (30 micromol/l) or nonspecific LOX inhibitor (NDGA, 0.1 mmol/l) which were administered separately or in combination. CF varied in autoregulatory range from 4.12+/-0.26 ml/min/g wt at 50 cm H2O to 5.22+/-0.26 ml/min/g wt at 90 cm H2O. In autoregulatory range, nitrite outflow varied from 2.05+/-0.17 nmol/min/g wt at 50 cm H2O to 2.52+/-0.21 nmol/min/g wt at 90 cm H2O and was strictly parallel with CPP/CF curve. The autoregulatory range of CF was significantly extended (40-100 cm H2O, 2.22+/-0.12 ml/min/g wt and 2.90+/-0.25 ml/min/g wt, respectively) under the influence of L-NAME. Hemodynamic effects were accompanied by significant decrease in nitrite outflow after L-NAME administration (0.56+/-0.11 nmol/min/g wt at 40 cm H2O to 1.45+/-0.14 nmol/min/g wt at 100 cm H2O). NDGA affected CF in the range of CPP 40-70 cm H2O only (from 42% at 50 cm H2O to 12% at 90 cm H2O, respectively) with no significant changes in nitrite outflow. When L-NAME was applied in combination with NDGA vs. NDGA only, CF was significantly reduced (from 34% at 50 cm H2O to 50% at 90 cm H2O, respectively) with parallel changes in nitrite outflow (from 40% at 50 cm H2O to 51% at 90 cm H2O, respectively). The results showed that CF and nitrite outflow could be decreased under L-NAME administration. Nonselective LOX inhibitor (NDGA) decreased control values of CF only at lower values of CPP but did not change nitrite outflow indicating antioxidant properties of NDGA. In addition, L-NAME decreased the effects induced by NDGA on CF and nitrite outflow indicating the role of NO.     

Association of two genetic variations of lipoprotein lipase, S447X and Hind III, with coronary artery disease and hypertriglyceridemia

This study was performed to assess the effect of the S447X and Hind III lipoprotein lipase gene polymorphisms on development of coronary artery disease and hypertriglyceridemia. The study included 132 patients and 98 healthy control subjects of Croatian descent. The lipoprotein lipase S447X polymorphism was associated with coronary artery disease and hypertriglyceridemia, as indicated by the lower frequency of S447 allele in the patient group (p = 0.005) and odds ratio (O.R = 0.40, p = 0.006). The patient and control groups also showed a significant difference in the distribution of Hind III/S447X genotype combinations (p = 0.013). There were no significant associations with lipid parameters for any genotype or genotype combination in the patient group. Frequencies of the S447X polymorphism and S447X/Hind III combinations differed between the CAD/TG and control group, thus these polymorphisms may be associated with CAD and hypertriglyceridemia.     

Regulated secretion of a protease activates intercellular signaling during fruiting body formation in M. xanthus

In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. There are two morphogenetic events in this program, aggregation and sporulation, which are temporally and spatially coordinated by the contact-dependent intercellular C-signal protein (p17). p17 is generated by proteolytic cleavage of the p25 precursor protein, which accumulates in the outer membrane of vegetative and starving cells. However, p17 generation is restricted to starving cells. Here we identify the subtilisin-like protease PopC that is directly responsible for cleavage of p25. PopC accumulates in the cytoplasm of vegetative cells but is selectively secreted during starvation coinciding with the generation of p17. Consequently, p25 and PopC only encounter each other in starving cells. Thus, restriction of p25 cleavage to starving cells occurs by a compartmentalization mechanism that depends on selective secretion of PopC during starvation. Our results provide evidence for regulated proteolysis via regulated secretion.     

Feedback dynamics and cell function: Why systems biology is called Systems Biology

A new paradigm, like Systems Biology, should challenge the way research has been conducted previously. This Opinion article aims to present Systems Biology, not as the application of engineering principles to biology but as a merger of systems- and control theory with molecular- and cell biology. In our view, the central dogma of Systems Biology is that it is system dynamics that gives rise to the functioning and function of cells. The concepts of feedback regulation and control of pathways and the coordination of cell function are emphasized as an important area of Systems Biology research. The hurdles and risks for this area are discussed from the perspective of dynamic pathway modelling. Most of all, the aim of this article is to promote mathematical modelling and simulation as a part of molecular- and cell biology. Systems Biology is a success if it is widely accepted that there is nothing more practical than a good theory.     

Interleukin-6 as possible early marker of stress response after femoral fracture

Toll-like receptor 4 (TLR4) activation is a key contributor to the carcinogenesis of colon cancer. Overexpression of galectin-1 (Gal-1) also correlates with increased invasive activity of colorectal cancer. Lactate production is a critical predictive factor of risk of metastasis, but the functional relationship between intracellular lactate and Gal-1 expression in TLR4-activated colon cancer remains unknown. In this study, we investigated the underlying mechanism and role of Gal-1 in metastasis and invasion of colorectal cancer (CRC) cells after TLR4 stimulation. Exposure to the TLR4 ligand lipopolysaccharide (LPS) increased expression of Gal-1, induced EMT-related cytokines, triggered the activation of glycolysis-related enzymes, and promoted lactate production. Gene silencing of TLR4 and Gal-1 in CRC cells inhibited lactate-mediated epithelial-mesenchymal transition (EMT) after TLR4 stimulation. Gal-1-mediated activation of a disintegrin and metalloproteinase 10 (ADAM10) and ADAM 17 increased the invasion activity and expression of mesenchymal characteristics in LPS-activated CRC cells. Conversely, inhibition of ADAM10 or ADAM17 effectively blocked the generation of lactate and the migration capacity of LPS-treated CRC cells. Thus, the TLR4/Gal-1 signaling pathway regulates lactate-mediated EMT processes through the activation of ADAM10 and ADAM17 in CRC cells.     

The influence of acclimatization on stress hormone concentration in serum during heat stress

This study was aimed to examine the influence of acclimatization on the change of concentration of stress hormones in men’s serum exposed to heat stress during physical training. The study included a total of 40 men, aged 19–21 years, divided randomly into four groups: CTRL group: control, exposed to the Exercise Tolerance Testing in comfortable conditions; O group: exposed to Exercise Tolerance Testing in a warm environment; P group: exposed to passive acclimation to heat for 10 days, followed by Exercise Tolerance Testing in a warm environment; A group: exposed to active acclimation to heat for 10 days, followed by Exercise Tolerance Testing in a warm environment. All participants were tested for thermoregulation and acclimatization, skin and tympanic temperature, heart rate (HR), hormonal status and sweating. The mean skin temperature was the lowest in the control group of subjects exposed to physical exertion under comfortable conditions, and at each point of measurement it was statistically significantly different from that of the other study groups (p?<?0.001). Sweating intensity was statistically significantly the lowest in the CTRL group (0.32?±?0.04 l/m²/h; p?<?0.001), compared to all other groups. Cortisol was significantly altered in O group (632.2?±?92.3; 467.2?±?89.7), testosterone levels were significantly altered in P (19.2?±?9.3; 16.4?±?7.3) and in A groups (22.1?±?12.4; 14.9?±?9.9), while prolactin was changed in O (392.1?±?51.3; 181.4?±?42.3), P (595.1?±?191.1; 191.2?±?52.5), and A group (407.4?±?189.3; 173.4?±?43.9) after the experimental period. The impact of acclimatization on hormonal indicators emphasizes its importance in the response of the endocrine system of soldiers to perform military activities in warm climates.

     

Ribosome Assembly Factors Pwp1 and Nop12 Are Important for Folding of 5.8S rRNA during Ribosome Biogenesis in Saccharomyces cerevisiae

Previous work from our lab suggests that a group of interdependent assembly factors (A₃ factors) is necessary to create early, stable preribosomes. Many of these proteins bind at or near internal transcribed spacer 2 (ITS2), but in their absence, ITS1 is not removed from rRNA, suggesting long-range communication between these two spacers. By comparing the nonessential assembly factors Nop12 and Pwp1, we show that misfolding of rRNA is sufficient to perturb early steps of biogenesis, but it is the lack of A₃ factors that results in turnover of early preribosomes. Deletion of NOP12 significantly inhibits 27SA₃ pre-rRNA processing, even though the A₃ factors are present in preribosomes. Furthermore, pre-rRNAs are stable, indicating that the block in processing is not sufficient to trigger turnover. This is in contrast to the absence of Pwp1, in which the A₃ factors are not present and pre-rRNAs are unstable. In vivo RNA structure probing revealed that the pre-rRNA processing defects are due to misfolding of 5.8S rRNA. In the absence of Nop12 and Pwp1, rRNA helix 5 is not stably formed. Interestingly, the absence of Nop12 results in the formation of an alternative yet unproductive helix 5 when cells are grown at low temperatures.     

Deoxyribonuclease I Inhibitory Properties,Molecular Docking and Molecular Dynamics Simulations of 1-(Pyrrolidin-2-yl)propan-2-one Derivatives

Deoxyribonuclease I (DNase I) inhibitory properties of two 1-(pyrrolidin-2-yl)propan-2-one derivatives were examined in vitro. Determined IC₅₀ values of 1-[1-(4-methoxyphenyl)pyrrolidin-2-yl]propan-2-one ( 1 ) (192.13±16.95 μM) and 1-[1-(3,4,5-trimethoxyphenyl)pyrrolidin-2-yl]propan-2-one ( 2 ) (132.62±9.92 μM) exceed IC₅₀ value of crystal violet, used as a positive control, 1.89- and 2.73-times, respectively. Compounds are predicted to be nontoxic and to have favorable pharmacokinetic profiles, with high gastrointestinal absorption and blood-brain barrier permeability. Molecular docking and molecular dynamics simulations suggest that interactions with Glu 39, Glu 78, Arg 111, Pro 137, Asp 251 and His 252 are an important factor for inhibitors affinity toward the DNase I. Determined inhibitory properties along with predicted ADMET profiles and observed interactions would be beneficial for the discovery of new active 1-(pyrrolidin-2-yl)propan-2-one-based inhibitors of DNase I.     

Role of the yeast Rrp1 protein in the dynamics of pre-ribosome maturation

The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA(3) to 27SB(S) pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA(3) and 27SB(L) pre-ribosomal RNAs.