MEK6 regulates human involucrin gene expression via a p38alpha - and p38delta -dependent mechanism.

A signaling cascade that includes protein kinase C (PKC), Ras, and MEKK1 regulates involucrin (hINV) gene expression in epidermal keratinocytes (Efimova, T., LaCelle, P., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395 and Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). Because signal transfer downstream of MEKK1 may involve several MAPK kinases (MEKs), it is important to evaluate the regulatory role of each MEK isoform. In the present study we evaluate the role of MEK6 in transmitting this signal. Constitutively active MEK6 (caMEK6) increases hINV promoter activity and increases endogenous hINV levels. The caMEK6-dependent increase in gene expression is inhibited by the p38 MAPK inhibitor, SB203580, and is associated with a marked increase in p38alpha MAPK activity; JNK and ERK kinases are not activated. In addition, hINV gene expression is inhibited by dominant-negative p38alpha and increased when caMEK6 and p38alpha are co-expressed. caMEK6 also activates p38delta, but p38delta inhibits the caMEK6-dependent activation. These results suggest that MEK6 increases hINV gene expression by regulating the balance between activation of p38alpha, which increases gene expression, and p38delta, which decreases gene expression.

MEK7-dependent activation of p38 MAP kinase in keratinocytes.

Previous studies suggest that a PKC/Ras/MEKK1 cascade regulates involucrin (hINV) gene expression in human epidermal keratinocytes. MEK7, which is expressed in epidermis, has been identified as a member of this cascade (Efimova, T., LaCelle, P., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395 and Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). However, the kinase that functions downstream of MEK7 has not been identified. Our present studies show that MEK7 expression in keratinocytes markedly activates p38alpha and modestly activates JNK. Activation of p38 MAPK by MEK7 is a novel finding, as previous reports have assigned MEK7 as a JNK regulator. We also demonstrate that this regulation is physiologically important, as the p38alpha- and JNK-dependent activities regulate hINV promoter activity and expression of the endogenous hINV gene.

Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein alpha (C/EBPalpha) DNA binding.

Human involucrin (hINV) is a keratinocyte differentiation marker expressed in the suprabasal epidermal layers. In cultured keratinocytes hINV mRNA levels are increased 10-fold by a 24-h treatment with 50 ng/ml PMA, an agent that promotes keratinocyte differentiation. Previous studies show that thapsigargin (TGN), an agent that depletes intracellular calcium stores, inhibits keratinocyte differentiation. In the present study we show that TGN inhibits the PMA-dependent, differentiation-associated, increase in hINV mRNA levels and hINV promoter activity. Inhibition is half-maximal at 10 nM and maximal at 100 nM TGN. Neither basal hINV promoter activity nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels are inhibited. Mutation of a functionally important CAATT-enhancer-binding protein (C/EBP) site within the hINV promoter proximal regulatory region eliminates the regulation, suggesting that TGN may effect C/EBP-dependent promoter activation. Consistent with this hypothesis, TGN inhibits C/EBPalpha-dependent promoter activation via a mechanism that involves inhibition of C/EBPalpha binding to DNA without changing C/EBPalpha protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.

Regulation of human involucrin promoter activity by novel protein kinase C isoforms.

Human involucrin (hINV) mRNA level and promoter activity increase when keratinocytes are treated with the differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). This response is mediated via a p38 mitogen-activated protein kinase-dependent pathway that targets activator protein 1 (Efimova, T., LaCelle, P. T. , Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395). In the present study we examine the role of various PKC isoforms in this regulation. Transfection of expression plasmids encoding the novel PKC isoforms delta, epsilon, and eta increase hINV promoter activity. In contrast, neither conventional PKC isoforms (alpha, beta, and gamma) nor the atypical isoform (zeta) regulate promoter activity. Consistent with these observations, promoter activity is inhibited by the PKCdelta-selective inhibitor, rottlerin, but not by Go-6976, an inhibitor of conventional PKC isoforms, and novel PKC isoform-dependent promoter activation is inhibited by dominant-negative PKCdelta. This regulation appears to be physiologically important, as transfection of keratinocytes with PKCdelta, -epsilon, or -eta increases expression of the endogenous hINV gene. Synergistic promoter activation (>/=100-fold) is observed when PKCepsilon- or -eta-transfected cells are treated with TPA. In contrast, the PKCdelta-dependent response is more complex as either activation or inhibition is observed, depending upon PKCdelta concentration.

Insulin-like growth factor binding protein-3 does not mediate the interferon-dependent suppression of human ectocervical epithelial cell proliferation.

Interferon is a potential therapeutic agent for the treatment of cervical cancer. In the present study we examine the role of IFNgamma as a regulator of proliferation and production of IGFBP-3 expression in ectocervical epithelial cells. ECE16-1 cells are a model for studying early human papillomavirus-dependent cervical disease. IFNgamma produces a concentration-dependent inhibition of ECE16-1 cell proliferation that is associated with an increase in insulin-like growth factor binding protein-3 level. Growth suppression and IGFBP-3 increase is maximal at concentrations of IFNgamma >/=0.75 ng/ml. The increased IGFBP-3 expression is mediated via an increase in IGFBP-3 encoding mRNA. In contrast, IFNgamma inhibits proliferation of CaSki and SiHa cells, but IGFBP-3 is barely detectable and levels are not regulated by IFNgamma. These results suggest that the IFNgamma-dependent suppression of CaSki and SiHa cell proliferation is not mediated by secreted IGFBP-3. This result was confirmed when vector-mediated overexpression of immunoreactive IGFBP-3 in SiHa and CaSki cells did not consistently result in reduced cell proliferation rate.

CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of keratinocyte differentiation and of involucrin gene expression. In the present study we show that a CCAAT/enhancer-binding protein (C/EBP) site in the proximal regulatory region is required for the phorbol ester response. Mutation of the C/EBP site results in the loss of basal and TPA-responsive activity. Gel mobility supershift analysis shows that C/EBPalpha binding to this site is increased by TPA treatment. Moreover, cotransfection of the human involucrin reporter plasmid with C/EBPalpha increases promoter activity to an extent comparable with TPA treatment. Mutation of the C/EBP-binding site eliminates these responses. Transfection experiments using GADD153 to create C/EBP-null conditions confirm that C/EBP factors are absolutely required for promoter activity and TPA responsiveness. C/EBPbeta and C/EBPdelta inhibit both TPA- and C/EBPalpha-dependent promoter activation, indicating functional differences among C/EBP family members. These results suggest that C/EBP transcription factor activity is necessary for basal promoter activity and TPA response of the involucrin gene.

Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.

Involucrin is a marker of keratinocyte terminal differentiation. Our previous studies show that involucrin mRNA levels are increased by the keratinocyte differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614-12622). We now study the signaling cascade responsible for this regulation. Protein kinase C and tyrosine kinase inhibitors inhibit both the TPA-dependent mRNA increase and the TPA-dependent increase in hINV promoter activity. The relevant response element is located within the promoter proximal regulatory region and includes an AP1 site, AP1-1. Co-transfection of the hINV promoter with dominant negative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the activity can be inhibited by dominant negative MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in activity and the dominant negative forms of these kinases failed to suppress TPA-dependent transcription. Treatment with an S6 kinase (S6K) inhibitor, or transfection with constitutively active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes protein kinase C, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also may function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.

[Negative potentials of the human brain to visual stimuli evoking saccades]. / Negativnye potentsialy golovnogo mozga cheloveka na zritel'nye stimuly, vyzyvaiushchie sakkady.

EEG of the frontal and parietal areas (F3, F4, Fz, P3, P4 according to the International 10-20 System) was recorded in 4 healthy subjects aged 20-25 years. The saccades were elicited by the shift of the light stimulus to the peripheral diodes (step or gap stimulation scheme). A complex fast event-related potential was evoked by the visual stimulation prior to the saccade onset. Amplitudes and topography of this potential depended on the saccade latency, stimulation scheme, spatial pattern of stimuli presentation and individual features of a subject. It is suggested that the early negative potentials reflect the processes of sensory processing of the stimuli spatial patterns and perceptive attention. The negative potentials with the intermediate latency may reflect the processes of activation of the cortical structures which participate in programming and triggering the saccades.

[Sorption of enzymes on ion exchange resins of various structure]. / Sorbtsiia fermentov na ionitakh razlichnoi struktury.

The isothermic adsorption of microbial and animal enzymes on carboxyl and sulpha-cation exchange resins was studied. The adsorption isotherms are curves with a maximum. The adsorption of alpha-amylase was studied in the presence of organic solvents. It was found that organic solvents influenced the isothermic adsorption of alpha-amylase, which is associated with changes in the interactions between protein molecules in solution. The adsorption system was in equilibrium in all the cases.

[The efficacy of muscle electrostimulation in the combined treatment of children with congenital cleft palate]. / Effektivnost' élektromiostimuliatsii v kompleksnom lechenii detei s vrozhdennoi rasshchelinoi neba.

The technique of electrostimulation of the soft palate muscles of children with cleft palate is described. Low-frequency electrostimulation courses were carried out before and after surgery in accordance with the degree of decompensation of the palatopharyngeal junction. This method was effectively used in 30 children with cleft palate. The treatment efficacy was assessed by the morphometric and electromyographic methods, that have confirmed that electrostimulation improved the morphology of soft palatal muscles.

[Structure of interstitial nephritis in children]. / Struktura interstitsial'nogo nefrita u detei.

The authors provide the data on 24 cases of interstitial nephritis in children treated at the Nephrological Center of the city of Kuibyshev in 1986-1990. Describe the etiological structure of interstitial nephritis using the classification developed by N. A. Korovina and coworkers (1982), randomization according to the age, sex, and the disease course. Demonstrate the predominance of the postviral and toxicoallergic disease patterns in children of the early, preschool and junior school age. Estimate the importance of the types of urine proteinograms in the diagnosis of interstitial nephritis.

[Effects of lidocaine on intramural circulation in isolated rabbit heart ventricle preparations]. / Deistvie lidokaina na intramural'nuiu tsirkuliatsiiu v izolirovannykh zheludochkovykh preparatakh serdtsa krolika.

Mapping was used on isolated rabbit ventricular specimens to study effects of lidocaine, 2-8 mg/l, on persistent intramural reentry involving the areas of slow transmural conduction. The agent was shown to produce antiarrhythmic and arrhythmogenic effects at the same time. Lidocaine reduced the duration of an arrhythmia, but provoked its initiation. Both these effects of lidocaine were attributable to its action on the refractory period of a slow transmural conduction area.

[Cytophysiology of Penicillium solitum: a producer of lipase]. / Tsitofiziologiia Penicillium solitum--produtsenta lipazy.

The cell population of Penicillin solitum was studied during maximum accumulation of lipase in the medium with electron microscopic and immunofluorescence methods. The data provided a conclusion that 2 types of lypolytic enzymes with various substrate and antigenic characteristics formed in the cells of P. solitum. It is likely that there is a specific inductor for exolipase synthesis as well as relationship endoenzymatic systems.

[The effect of lipase from Penicillium sp. on the membrane fraction of the levorin-producing Streptomyces levoris]. / Deistvie lipazy Penicillium sp. na membrannuiu fraktsiiu produtsenta levorina Streptomyces levoris.

The effect of a lipase preparation from Penicillium sp. on the membranes of the levorin producer Streptomyces levoris was being studied. The enzyme preparation was found preferably to hydrolyse neutral lipids in the Str. levoris membranes, which makes it possible to use the lipase from Penicillium sp. for studying neutral lipids in microbial membranes.

[Biosynthesis of the polyene antibiotic levorin and the membrane composition of Streptomyces levoris]. / Biosintez polienovogo antibiotika levorina i sostav membran Streptomyces levoris.

Changes in the composition of Streptomyces levoris membranes were studied in the course of the polyene antibiotic levorin biosynthesis when the process was stimulated by Candida tropicalis metabolites and inhibited by inorganic phosphate. In the presence of stimulating compounds, the percentage of membranes increased in S. levoris cells and the membrane composition changed: the protein-to-lipid ratio and the concentration of total phosphorus decreased while the content of carbohydrates increased. Analysis of the lipid component showed that these changes were due to a gradual substitution of non-phosphorus glycolipids for membrane phospholipids and to an enrichment of the membranes with proteolipids. Such changes were not detected in a medium containing the inhibitor. It was for the first time that a considerable amount of the antibiotic produced by the culture was found in the membrane fractions. The data are discussed in relation with a possible role of the actinomycete membrane structures in levorin biosynthesis.

[Effect of antibiotics on D-serotonin-reactive structures]. / Vliianie nekotorykh antibiotikov na D-serotoninoreaktivnye struktury.

Experiments on the isolated organs showed that ampicillin and levomycetin have pronounced D-antiserotoninergic effects; antagonism of antibodies and serotonin was found to be of competitive type. At an increase in levomycetin dosage D-antiserotoninergic effect was followed by the spasmolytic effect. Kefzol and benzylpenicillin failed to show any D-antiserotonin-ergic properties.

[Action of the products of the vital activity of yeastlike fungi on the biosynthesis of levorin, levoristatin and fatty acids by a Streptomyces levoris culture]. / Deistvie produktov zhizndeiatel'nosti drozhzhepodobnykh gribov na biosintez levorina, levoristatina i zhirnykh kislot kul'turoi Streptomyces levoris.

The data on the effect of the products of vital activity of Candida tropicalis, a yeast-like fungus, on the biosynthesis of levorin, levoristatin and fatty acids by Streptomyces levoris are presented. It was shown that the effect of the biostimulators was not specific with respect to production of levorin, since in the presence of the products of vital activity of C. tropicalis an increase in the synthesis of levoristatin and fatty acids was also observed. The qualitative and quantitative composition of the fatty acids of the mycelium of S. levoris was studied. Interrelation between the biosynthesis of levorin and synthesis of unsaturated fatty acids and branched chain fatty acids was noted.