Efficient apoptosis and necrosis induction by proteasome inhibitor: bortezomib in the DLD-1 human colon cancer cell line

The inhibition of the 26S proteasome evokes endoplasmic reticulum stress, which has been shown to be implicated in the antitumoral effects of proteasome inhibitors. The cellular and molecular effects of the proteasome inhibitor—bortezomib—on human colon cancer cells are as yet poorly characterized. Bortezomib selectively induces apoptosis in some cancer cells. However, the nature of its selectivity remains unknown. Previously, we demonstrated that, in contrast to normal fibroblasts, bortezomib treatment evoked strong effect on apoptosis of breast cancer cells incubated in hypoxic and normoxic conditions. The study presented here provides novel information on the cellular effects of bortezomib in DLD-1 colon cancer cells line. We observe twofold higher percentage of apoptotic cells incubated for 48 h with 25 and 50 nmol/l of bortezomib in hypoxic conditions and four-, fivefold increase in normoxic conditions in comparison to control cells, incubated without bortezomib. It is of interest that bortezomib evokes strong effect on necrosis of DLD-1 colon cancer cell line. We observe the sixfold increase in necrosis of DLD-1 cells incubated with 25 or 50 nmol/l of bortezomib for 48 h in hypoxia and fourfold increase in normoxic conditions in comparison to adequate controls. We suggest that bortezomib may be candidates for further evaluation as chemotherapeutic agents for human colon cancer.     

The effect of hypoxia on the expression of 150 kDa oxygen-regulated protein (ORP 150) in HeLa cells.

Correct protein folding is an important factor, for the translocation of newly synthesised proteins to specific subcellular compartments, extracellular matrix or to biological fluids. This process is regulated by a group of specific proteins, referred to as chaperones. Many stress conditions, such as oxygen or glucose deprivation, slow down the folding process and cause accumulation of unfolded/misfolded proteins in the cell. Molecular chaperones are induced in these conditions; with some named as oxygen-regulated proteins (ORPs). These bind to unfolded / misfolded proteins to facilitate correct assembly. ORP 150 is the subject of this study. Hypoxia results in an enhancement of ORP 150 expression in several tumour cell lines cultured in vitro. HeLa cells grown in hypoxic conditions (despite an intensive expression of ORP 150) demonstrate higher rates of apoptosis in comparison to those cultured in normoxic conditions. Furthermore, the inhibition of ORP 150 synthesis by transfection of these cells with a specific siRNA resulted in an intensification of apoptosis, as indicated by specific markers of this process; the enhancement of poly ADP-ribose protein cleavage and the increase in Bim protein expression. We conclude from our study that the increase in ORP 150 synthesis protects the cells against the proapoptotic effect of hypoxia.     

Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation

Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA^Gln formation. To analyze the peculiarities of the structure–function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3Å and a docking model of the synthetase complexed to tRNA^Gln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA^Gln are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA^Gln recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA^Gln acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.     

Tissue fluid pressure and flow during pneumatic compression in lymphedema of lower limbs

Background: Physiotherapy of edema in cases with obstructed main lymphatics of lower limbs requires knowledge of how high external pressures should be applied manually or set in compression devices in order to generate tissue pressures high enough to move tissue fluid to nonswollen regions and to measure its flow rate. Methods: We measured tissue fluid pressure and flow in subcutaneous tissue of lymphedematous limbs stages II to IV at rest and during pneumatic compression under various pressures and inflation timing. An 8-chamber sequential compression device inflated to pressures 50-120?mmHg, for 50 sec each chamber, with no distal deflation, was used. Pressures were measured using a wick-in-needle and electronic manometer. Fluid flow was calculated from continuously recorded changes in limb circumference using strain gauge plethysmography. Results: Before massage, in all stages of lymphedema, stagnant tissue fluid pressures in subcutaneous tissue ranged between -1 and +10 mmHg and did not differ from those measured in normal subjects. Pressures generated in tissue fluid by pneumatic compression reached 40-100 mmHg and were lower than those in inflated chambers. High pressure gradient through the skin was caused by its rigidity (fibrosis) and dissipation of applied compression force to proximal noncompressed limb regions. The calculated volumes of displaced tissue fluid ranged from 10 to 30 ml per compression cycle, to reach in some cases 100 ml in the groin region. Conclusions: Tissue fluid pressures generated by a pneumatic device were found lower than in the compression chambers. The obtained results point to the necessity of applying high pressures and longer compression times to generate effective tissue fluid pressures and to provide enough time for moving the stagnant fluid.     

Swapping of three-dimensional domains as a molecular mechanism of dimerization of aminoacyl-tRNA synthetases

It has been shown recently that many proteins undergo oligomerization through exchange of structural elements. That process, termed a 3D domain swapping, is the replacement of a portion of the tertiary structure of a protein with an identical piece from a second polypeptide chain. When the exchange is reciprocated, domain-swapped dimers embrace with the exchange of elements of secondary structure or domains; however, if the exchange is not reciprocated but propagated along multiple polynucleotide chains, higher-order assemblies may form. In this paper we discuss swapping as a general mechanism of aminoacyl-tRNA synthetases dimerization, specifically plant methionyl-tRNA synthetase.     

Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves

Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature.     

New derivatives of picolinic acid and nicotinic acid with anticonvulsant activity

Previously obtained Pic-BZA is a potent anticonvulsant with low neurotoxicity, but its half-time of action is only about 15 min. In search for equally effective anticonvulsants but with a longer time of action fourteen Pic-BZA analogs were obtained. The compounds were evaluated in the Anticonvulsant Screening Project (ASP) of Antiepileptic Drug Development Program (ADDP) of NIH. Picolinic acid 2-fluorobenzylamide (Pic-2-F-BZA, 7) appeared to be the most effective compound of the series.     

Limb lymph node response to bone fracture

In previous clinical studies, dilation of afferent lymphatics and enlargement of inguinal lymph nodes (LN) were observed in lymphoscintigrams from patients with persistent posttraumatic edema of lower extremities after fractures and trauma of soft tissues. In this study, changes in rat popliteal and iliac lymph nodes draining lymph from the site of tibial fracture and adjacent soft tissue injury were investigated. The observed parameters were lymph node weight, cell number, phenotype frequency, cell cytokine expression, and reactivity to mitogens. The key observations included: a) increase in the weight and total cell number of the lymph nodes; b) increased autotransformation rate and responsiveness of lymph node cells to mitogen; c) decreased frequency of ED1 macrophages and activated OX8 cytotoxic cells in flow cytometry analysis; d) high expression of OX6 class II-positive, OX7 (stem cells), OX62 (migrating dendritic cells), ED1 (macrophages), and OX12 (B cells) on immunohistochemical sections of LNs with some few HIS48 (granulocytes); e) high expression of NOS3 and TGF beta by lymph node lymphocytes and endothelial cells. In summary, local lymph nodes reacted to internal wounds, such as bone fracture and injury to adjacent tissues, through mobilization of cells from the blood circulation, along with activation of cellular subsets. The molecular mechanism that provides the signal for this reaction remains unknown. The absence of major changes in the frequency of lymph node cell subpopulations indicates that lymph nodes are constitutively prepared for influx of antigens from damaged tissues and react only with increase in cell number and cell activation. The nature of the reaction, including lack of immunization against autoantigens, remains unclear. Further elucidation will require studies on the mechanism of cross-tolerance to self-antigens during wound healing.     

Differential effect of fasting on IGF-BPs in serum of young and adult rats and its implication to impaired skin GAG content

During fasting or aging of animals there is a decreased content of skin glycosaminoglycans (GAGs). It has been found that the skin of adult rats contains about 60% of GAGs found in the skin of young animals. Fasting of both groups of animals (young and adult) resulted in decrease of GAG content. However, GAG content in the skin of fasted young rats decreased by 30% and in fasted adult rats by 15% only, compared to fed animals, respectively. The mechanism for the phenomena is not known. We considered insulin-like growth factor-I (IGF-I) as a potential candidate involved in regulation of GAG biosynthesis in both experimental models of animals. Adult rat sera were found to contain about 75% of IGF-I recovered from young rat sera. Fasting of both groups of animals resulted in dramatic decrease in serum IGF-I levels to about 50% of initial values. Since IGF-I activity and IGF-I serum half-life depends on the level of specific IGF-binding proteins (IGFBPs) we determined (i) relationship between main groups of IGFBPs, namely high molecular weight binding proteins (HMWBPs) and low molecular weight binding proteins (LMWBPs) and (ii) the amounts of IGF-I bound to respective proteins in the sera of all experimental animals. Control young rat serum was found to contain about 90% of HMWBPs and about 10% of LMWBPs as determined by ligand binding assay. In contrast, control adult rat serum contained about 60% of HMWBPs and about 40% of LMWBPs. Fasting of both groups of animals resulted in significant increase in serum levels of LMWBPs. Control young rat serum was found to contain about 8% IGF-I bound to LMWBPs while serum of control adult rats contained 18% IGF-I bound to these proteins. In sera of fasted young animals however, about 75% of the bound IGF-I was recovered from LMWBPs (about 60% of total serum IGF-I) while in sera of fasted adult animals only about 56% of the bound IGF-I was recovered from LMWBPs (about 50% of total serum IGF-I). Evidence was provided that during fasting of both groups of animals there is a significant decrease in serum BP-3 and dramatic increase in serum BP-1 concentrations, compared to respective controls. However, the concentration of BP-1 in serum of fasted young rats was increased by about 60 fold while in serum of fasted adult rats only by about 10 fold, compared to respective control animals. Negative correlation between skin GAG content and LMWBPs derived IGF-I during fasting of young (r = - 0.943, p < 0.001) and adult ( r = - 0.571, p < 0.01) rats was found.The data presented suggest that the effects of aging and fasting on decreased skin GAG content may be due to induction of LMWBPs that are known to (i) inhibit IGF-I dependent function and (ii) increase clearance of IGF-I from circulation. However, the effects of fasting are distinct in respect to young and adult rats suggesting that mechanisms involved in regulation of IGF-I bioactivity during aging are more complex that during fasting.