Depletion of COPI in cancer cells: the role of reactive oxygen species in the induction of lipid accumulation, noncanonical lipophagy and apoptosis.

The coatomer protein complex 1 (COPI) is a multisubunit complex that coats intracellular vesicles and is involved in intracellular protein trafficking. Recently we and others found that depletion of COPI complex subunits zeta (COPZ1) and delta (ARCN1) preferentially kills tumor cells relative to normal cells. Here we delineate the specific cellular effects and sequence of events of COPI complex depletion in tumor cells. We find that this depletion leads to the inhibition of mitochondrial oxidative phosphorylation and the elevation of reactive oxygen species (ROS) production, followed by accumulation of lipid droplets (LDs) and autophagy-associated proteins LC3-II and SQSTM1/p62 and, finally, apoptosis of the tumor cells. Inactivation of ROS in COPI-depleted cells with the mitochondrial-specific quencher, mitoquinone mesylate, attenuated apoptosis and markedly decreased both the size and the number of LDs. COPI depletion caused ROS-dependent accumulation of LC3-II and SQSTM1 which colocalizes with LDs. Lack of double-membrane autophagosomes and insensitivity to Atg5 deletion suggested an accumulation of a microlipophagy complex on the surface of LDs induced by depletion of the COPI complex. Our findings suggest a sequence of cellular events triggered by COPI depletion, starting with inhibition of oxidative phosphorylation, followed by ROS activation and accumulation of LDs and apoptosis.

JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1.

BH3 interacting-domain death agonist (Bid) is a BH3-only pro-apoptotic member of the Bcl-2 family of proteins. Its function in apoptosis is associated with the proteolytic cleavage to the truncated form tBid, mainly by caspase-8. tBid translocates to mitochondria and assists Bax and Bak in induction of apoptosis. c-Jun N-terminal kinase (JNK)-dependent alternative processing of Bid to jBid was also reported. We have previously shown that the folate stress enzyme 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) activates JNK1 and JNK2 in cancer cells as a pro-apoptotic response. Here we report that in PC-3 prostate cancer cells, JNK1/2 phosphorylate Bid at Thr59 within the caspase cleavage site in response to ALDH1L1. In vitro, all three JNK isoforms, JNK 1-3, phosphorylated Thr59 of Bid with JNK1 being the least active. Thr59 phosphorylation protected Bid from cleavage by caspase-8, resulting in strong accumulation of the full-length protein and its translocation to mitochondria. Interestingly, although we did not observe jBid in response to ALDH1L1 in PC-3 cells, transient expression of Bid mutants lacking the caspase-8 cleavage site resulted in strong accumulation of jBid. Of note, a T59D mutant mimicking constitutive phosphorylation revealed more profound cleavage of Bid to jBid. JNK-driven Bid accumulation had a pro-apoptotic effect in our study: small interfering RNA silencing of either JNK1/2 or Bid prevented Bid phosphorylation and accumulation, and rescued ALDH1L1-expressing cells. As full-length Bid is a weaker apoptogen than tBid, we propose that the phosphorylation of Bid by JNKs, followed by the accumulation of the full-length protein, delays attainment of apoptosis, and allows the cell to evaluate the stress and make a decision regarding the response strategy. This mechanism perhaps can be modified by the alternative cleavage of phospho-T59 Bid to jBid at some conditions.

ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A.

Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

[The regulatory effect of mexidol on the hemoglobin level under acute stress conditions].

Experiments on a group of 39 Wistar albino male rats showed that, 2 and 5 days after a model acute stress procedure, the total amount of erythrocytes is increased. This process is accompanied by a hypochromia of red cells and by a decrease in the concentration of serum iron. Single administration of mexidol (100 mg/kg) before stress induction prevents changes in the amount of blood erythrocytes, as well as in their saturation with hemoglobin. Mexidol also prevents a decrease in the level of serum iron. The drug does not influence the hematological parameters and iron concentration in blood serum of intact animals 2 and 5 days after injection.

Cooperation between JNK1 and JNK2 in activation of p53 apoptotic pathway.

FDH (10-formyltetrahydrofolate dehydrogenase) is strongly downregulated in tumors while its elevation suppresses proliferation of cancer cells and induces p53-dependent apoptosis. We have previously shown that FDH induces phosphorylation of p53 at Ser6, which is a required step in the activation of apoptosis. In the present study, we report that FDH-induced p53 phosphorylation is carried out by JNK1 and JNK2 (c-Jun N-terminal kinases) working in concert. We have demonstrated that FDH induces phosphorylation of JNK1 and JNK2, while treatment of FDH-expressing cells with JNK inhibitor SP600125, as well as knockdown of JNK1 or JNK2 by siRNA, prevents phosphorylation of p53 at Ser6 and protects cells from apoptosis. Interestingly, the knockdown of JNK1 abolished phosphorylation of JNK2 in response to FDH, while knockdown of JNK2 did not prevent JNK1 phosphorylation. Pull-down assay with the p53-specific antibody has shown that JNK2, but not JNK1, is physically associated with p53. Our studies revealed a novel mechanism in which phosphorylation of JNK2 is mediated by JNK1 before phosphorylation of p53, and then p53 is directly phosphorylated by JNK2 at Ser6.

[Analysis of causes of unsatisfactory results of rectocele surgical treatment and ways of their correction]. / Analiz prichin neudovletvoritel'nykh rezul'tatov khirurgicheskogo lecheniia rektotsele i puti ikh ustraneniia.

Causes of recurrences and unsatisfactory results of rectocele surgical treatment (except wound suppuration after surgery) were analyzed. It was concluded that prolapse of rectal mucosa and (or) severe muscular atrophy of the pelvic fundus uncorrected during transvaginal correction of rectocele are the main causes. It is recommended to resect mucosal excess by a traditional or A. Longo's method in the former case and to use synthetic net in the latter. This brings about favorable results in these patients.

[On some peculiarities of stab wounds]. / O nekotorykh osobennostiakh ran, prichinennykh koliushchimi predmetami.

The article deals with the differential diagnosis of stab injuries inflicted upon by cylindrical and conic objects with a round cross-section and with a point-shaped and cross-shaped stings as well as by sharp three-edged objects. An analysis of the obtained data leads to the conclusion on a possibility of carrying out the group identification of the mentioned stab objects according to morphological specific features of injuries.

[The stress-protective effect of a new derivative of n-3-polyunsaturated fatty acids]. / Stress-protektornyi éffekt novogo proizvodnogo n-3-polinenasyshchennykh zhirnykh kislot.

It has been established that prophylactic oral administration of the new derivative of n-3 polyunsaturated fatty acids--P-55 in a daily dose of 0.2 g/kg during 30 days prevents some morphological and physiological manifestations of the chronic stress-syndrome in white rats. There were normalized body and some internal organs weights, content and distribution of ascorbic acid in the adrenal tissue; decreased intensity of gastric ulcerogenesis. The behaviour of animals became more quiet. It is concluded that the preparation P-55 has a stress-protective effect during its prophylactic administration.

[Forensic medicine examinations of injuries inflicted with flat puncturing and cutting objects]. / Sudebno-meditsinskie issledovaniia povrezhdenii, prichinennykh ploskimi koliushche-rubiashchimi predmetami.

A total of 560 injuries of the skin and of clothes made of various textiles were examined. The injuries were inflicted with puncturing-and-cutting instruments with a flat section (e.g. chisel). Morphologic signs of the injury permit establishing the position of the instrument during the injury, the width of the cutting edge and thickness of the lateral surface of the instrument with which the injury was inflicted, as well as some other features of the instrument and the conditions of the injury infliction.

[The use of methyldioxyline for the prevention of local complications following hemorrhoidectomy]. / Primenenie metildioksidina dlia profilaktiki mestnykh oslozhnenii posle gemorroidéktomii.

The article discusses the results of clinico-laboratory study of the application of methyl dioxyline ointment after hemorrhoidectomy with complete restoration of the anal canal mucosa. It is shown that the ointment is a reliable measure for the prevention of postoperative purulent complications, it improves the reparative processes. Its use in the treatment of wounds developing as the result of excision of hemorrhoids shortens the term of in-patient treatment after the operation and the period of general incapacity.

[The role of the hypophysis and STH in the regulation of the androgenic receptor level of the rat liver and in the realization of the sex-differentiating action of gonadal hormones]. / Rol' gipofiza i STG v reguliatsii urovnia androgennykh retseptorov pecheni krys i v realizatsii poldifferentsiruiushchego deistviia gormonov gonad.

The role of the pituitary gland was investigated in the regulation of the level of rat liver androgenic receptors (AR) and in the realization of polydifferentiating action of gonadal hormones. The level of AR in the female and male rat liver was shown not to depend directly on the presence of the pituitary gland or STH administration. The pituitary gland is not a mediator in the realization of polydifferentiating action of androgens and estrogens. STH enhances the development of a positive effect of androgens on the level of AR.

[The characteristics of the sex-differentiating action of sex steroids on the androgen receptors of the rat liver]. / Osobennosti poldifferentsiruiushchego deistviia polovykh steroidov na retseptory androgenov pecheni krys.

A study was made of the specificities of polydifferentiating action of androgens (A) and estrogens (E) on the level of androgen receptors (AR) of the rat liver cytosol. Androgen receptors were shown to be detected in the neonatal period of ontogenesis, their level was progressively on the increase with the body development. Sex differences in their content were revealed in mature animals only. The AR level is a reversibly controlled, but unprogrammed sex-related sign. The effects of sex steroids are dose-related and modulated reciprocally by the action of endogenous A and E. The action of physiological doses of A and E is characterized by slow development of the effect. Correlation between the level of AR and the efficacy of A action on the content of these proteins was revealed.

[Demonstration of a sex differentiation in the content of androgen receptors in the liver cytosol of rats]. / Vyiavlenie polovoi differentsirovki v soderzhanii retseptorov androgenov v tsitozole pecheni krys.

The paper is concerned with differential quantification of the level of androgen receptors (AR) in the liver cytosol of male and female rats as well as in the prostatic cytosol. AR affinity to synthetic androgen R1881 was shown to be similar in all investigated tissues. In male rats the AR content in the liver cytosol was 20.5 +/- 2.1 fmol per 1 mg of protein, lower (p less than 0.05) than in the prostatic cytosol (30.8 +/- 2.5 fmol per 1 mg of protein). Sex differentiation of the liver AR content with 3-fold predominance in males was determined. Gonadectomy of mature rats caused leveling down of sex differences in the liver AR content as a result of a decrease in AR concentration in castrated males and its increase in ovariectomized females. Sex differentiation of the liver AR content could be an additional factor enhancing the multiple differentiating action of androgens on the male rat liver.

[Experience with the clinical use of lithium oxybutyrate in patients with cardiac rhythm disorders]. / Opyt klinicheskogo ispol'zovaniia litiia oksibutirata u bol'nykh s narusheniem ritma serdtsa.

An antiarrhythmic effect of lithium oxybutyrate was demonstrated in 81% of patients with ventricular extrasystole in the presence of affective disturbances, those with hypersympathicotonus in particular. In arrhythmic cases, lithium oxybutyrate was shown to have a hypokinetic effect on central hemodynamics and contribute to peripheral vasodilatation without causing clinically apparent circulatory disorders. The drug inhibits platelet aggregation and moderately increases blood procoagulation activity in the presence of greater erythrocyte deformability and improved bulbar conjunctival microcirculation.