Correlation among the BRAF gene mutation status, clinicopathological features of primary tumour, and lymph node metastasizing of papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumour. A common mutation of papillary thyroid carcinoma (PTC) is the somatic mutation of the BRAF (V600E) gene. AIM: The aim was to 1) determine the association of lymph node metastases of PTC with the BRAF gene mutation of primary tumour; 2) evaluate association of the BRAF mutation in the -primary tumour with clinicopathological para-meters; 3) examine the extent of genetic heterogeneity by monitoring the BRAF mutation in multicentric tumours. SUBJECTS AND METHODS: Retrospective analysis of the BRAF (V600E) mutation in PTC and PTC neck lymph node metastases in 156 patients operated from 2003 to 2012 in Prague and Zlín, the Czech Republic, using a qPCR assay. The results were correlated with clinicopathological factors. RESULTS: DNA was successfully extracted from 137 samples. The BRAF (V600E) mutation was detected in 78 cases (56.9%). The patients with BRAF p.Val600Glu mutation of primary tumour had only non-significantly higher risk of cervical lymph node metastases [OR=2.39 (95%) CI 1.00-5.75, p=0.052]. In the classic papillary variant, the BRAF (V600E) mutation was found significantly more often than in other PTC subtypes (p=0.022). We did not confirm any significant association between the BRAF (V600E) mutation and other clinicopathological findings. CONCLUSION: Except for the higher prevalence in papillary variant of PTC, BRAF p.Val600Glu mutation was not associated with other prognostic clinicopathological factors of PTC. BRAF mutation cannot be regarded as a reliable marker of node metastases in patients with PTC.

[Current trends in treatment and follow-up of patients with differentiated thyroid carcinoma - experience with the use of recombinant human thyrotropin]. / Soucasné trendy v lécbe a dispenzarizaci pacientu s diferencovaným karcinomem stítné zlázy - zkusenosti s vyuzitím rekombinantního humánního tyreotropinu.

Differentiated thyroid carcinoma is the most common endocrine malignancy with an excellent prognosis in the case of its early detection. Radioiodine 131I and thyroid hormones continue to be the pivotal drugs in treatment and follow-up for more than 50 years. The therapeutical and diagnostic options were recently expanded by the use of recombinant human thyrotropin (rhTSH). Our experience with the diagnostic administration of rhTSH confirms the outcomes of official trials and also indicates that the effect of rational therapy with 131I after rhTSH is similar to the outcome after standard regime using long-term thyroid hormone withdrawal.

Osmotin, a pathogenesis-related protein.

Plants protect themselves from pathogen invasion through the local expression of a variety of pathogenesis-related proteins. They are highly diverse in both primary structure and length, and exhibit different direct antimicrobial activity. This text reviews the knowledge of osmotin, antimicrobial protein involved in innate immunity of plants. Osmotin belongs to the fifth class of the group of pathogenesis-related (PR) proteins and has been found in different plants species, in every case osmotin is cysteine-rich protein involved in plant defense responses to several pathogens and abiotic stresses. The phylogenetic tree of amino acids compositions of osmotins from different plant species is presented and the basic similarities of clusters are discussed in this review. Osmotin gene is activated by different biotic as well as abiotic signals and has many functions. The review summarizes biochemical and structural properties, induction, functions and structural homology between osmotin and other proteins. Recent data about recombinant production in bacterial and plant cells are examined. The article indicates possible ways of osmotin application in research in the field of functional biology, medicine and agriculture.

Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity.

The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1-1.9 U mg(-1) dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30 degrees C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile >> chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92-100, 70-90 and 30-51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation.

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.

Proteolytic cleavage of carboxypeptidase N markedly increases its antifibrinolytic activity.

BACKGROUND: Carboxypeptidase N (CPN) is a constitutively active basic carboxypeptidase sharing specificity with activated thrombin-activable fibrinolysis inhibitor (TAFIa). Generally, CPN is regarded as being non-antifibrinolytic. However, this assumption has not been thoroughly investigated, particularly with respect to long-term antifibrinolysis. In addition, a recent report has shown that plasmin cleavage increases the catalytic activity of CPN. Therefore, we investigated the antifibrinolytic properties of CPN and plasmin-cleaved CPN (CPNc). METHODS: CPN was incubated with plasmin for various periods of time and the prolongation of clot lysis at various concentrations of CPN/CPNc mixture was investigated in TAFI-depleted plasma. CPN cleavage was analyzed by electrophoresis and catalytic activity was determined by monitoring cleavage of the small substrate, FA-Ala-Lys. RESULTS: CPN exhibited antifibrinolytic properties in plasma clot lysis assays when present at supraphysiological concentrations. Depletion of CPN from plasma decreased the lysis time of clots formed from minimally diluted plasma at low tissue-type plasminogen activator (t-PA) concentrations. Plasmin cleavage of CPN markedly increased the antifibrinolytic properties. CPN and CPNc prolonged lysis in a non-saturable, dose-dependent, and t-PA-dependent manner. At sufficient concentration, CPN and CPNc prolonged lysis at least forty-fivefold. CPNc was 700% more antifibrinolytic than CPN but only 7% more active toward FA-Ala-Lys. The active site inhibitor GEMSA eliminated the antifibrinolytic effects of CPN and CPNc. Antifibrinolytic activity correlated with cleavage of active and/or regulatory subunits, presumably generating heterodimeric CPNc. CONCLUSIONS: Limited proteolysis of CPN by plasmin generates an enzyme with greatly increased antifibrinolytic properties. We speculate that (patho)physiological proteolysis of CPN may generate a long-term antifibrinolytic enzyme.

Endogenous tumor necrosis factor alpha mediates enhanced apoptosis of cultured villous trophoblasts from intrauterine growth-restricted placentae.

Tumor necrosis factor alpha (TNFalpha) has been implicated in the abnormally high levels of trophoblast apoptosis seen in placentae from pregnancies complicated by small births. We examined the hypothesis that at physiological (35-50 mmHg) oxygen tensions, the production of TNFalpha stimulates the apoptosis of placental trophoblasts associated with infants that are intrauterine growth-restricted (IUGR). Highly purified cytotrophoblasts (CT) from IUGR-complicated pregnancies spontaneously underwent a higher rate of apoptosis after 24 h of culture at a normoxic (for villous CT) tension of 38 mmHg than did CT from normal placentae. Real-time PCR analysis of TNFalpha mRNA revealed approximately threefold higher levels in IUGR trophoblasts after culturing at a pO2 of 38 mmHg. A higher level of TNFalpha receptor p55 (which mediates apoptosis) was found in IUGR CT by western blot analysis at pO2 of <10, 38, and 140 mmHg. Neutralizing antibody to TNFalpha significantly inhibited the apoptosis of IUGR trophoblasts cultured at 38 mmHg and addition of TNFalpha significantly elevated apoptosis of normal and IUGR trophoblasts but less in IUGR cells cultured at <10 mmHg. We conclude that at physiological oxygen tensions (38 mmHg), villous CT from IUGR pregnancies, when compared with uncomplicated pregnancies, undergo more TNFalpha-induced apoptosis both because of elevated expression of TNFalpha and TNF receptor p55.

Multiple anti-apoptotic pathways stimulated by EGF in cytotrophoblasts.

Epidermal growth factor (EGF) reduces apoptosis in primary cytotrophoblast (CT) in culture through two separate pathways: the extracellular signal related kinase (ERK) 1/2 and phosphatidyl inositol 3-kinase (PI-3 kinase) paths. Whether other pathways are involved in survival signalling is unknown. We here show that the c-Jun NH2 terminal kinase (JNK) and the mitogen activated kinase (MAPK) p38 are also activated by EGF as seen by increases in JNK and p38 phosphorylation. However, inhibition of JNK phosphorylation with the specific inhibitor SP600125 increases apoptosis in a manner refractory to the addition of EGF but inhibition of p38 phosphorylation with its specific inhibitor SB 203580 does not increase apoptosis. EGF also activates sphingosine kinase-1 (SPHK-1), which converts sphingosine to sphingosine-1-phosphate, and its inhibition with dimethyl sphingosine (DMS) increased trophoblast death. Inhibition of SPHK-1 also did not affect EGF stimulated phosphorylation of PI-3 kinase, Akt, ERK1/2 or p38 but inhibition of PI-3 kinase with a specific inhibitor LY294002 partly (40%) inhibited the EGF-stimulated increase in SPHK-1 activity. We conclude that, in addition to the PI-3 kinase and ERK1/2 pathways, EGF acts through its receptor to stimulate JNK, p38 and SPHK-1 pathways, but that the JNK and SPHK-1, and not the p38, pathways are involved in suppressing apoptosis. This information provides evidence that EGF stimulates survival along multiple pathways that differ in trophoblast and other cell types.

Adrenergic regulation and diurnal rhythm of p38 mitogen-activated protein kinase phosphorylation in the rat pineal gland.

In this study, we investigated adrenergic and photoneural regulation of p38MAPK phosphorylation in the rat pineal gland. Norepinephrine (NE), the endogenous neurotransmitter, dose-dependently increased the levels of phosphorylated MAPK kinase 3/6 (MKK3/6) and p38MAPK in rat pinealocytes. Time-course studies showed a gradual increase in MKK3/6 and p38MAPK phosphorylation that peaked between 1 and 2 h and persisted for 4 h post NE stimulation. In cells treated with NE for 2 and 4 h, the inclusion of prazosin or propranolol reduced NE-induced MKK3/6 and p38MAPK phosphorylation, indicating involvement of both alpha- and beta-adrenergic receptors for the sustained response. Whereas treatment with dibutyryl cAMP or ionomycin mimicked the NE-induced MKK3/6 and p38MAPK phosphorylation, neither dibutyryl cGMP nor 4beta-phorbol 12-myristate 13-acetate had an effect. The NE-induced increase in MKK3/6 and p38MAPK phosphorylation was blocked by KT5720 (a protein kinase A inhibitor) and KN93 (a Ca(2+)/calmodulin-dependent kinase inhibitor), but not by KT5823 (a protein kinase G inhibitor) or calphostin C (a protein kinase C inhibitor). In animals housed under a lighting regimen with 12 h of light, MKK3/6 and p38MAPK phosphorylation increased in the rat pineal gland at zeitgeber time 18. The nocturnal increase in p38MAPK phosphorylation was blocked by exposing the animal to constant light and reduced by treatment with propranolol, a beta-adrenergic blocker. Together, our results indicate that activation of p38MAPK is under photoneural control in the rat pineal gland and that protein kinase A and intracellular Ca(2+) signaling pathways are involved in NE regulation of p38MAPK.

Ability of bacterial biphenyl dioxygenases from Burkholderia sp. LB400 and Comamonas testosteroni B-356 to catalyse oxygenation of ortho-hydroxychlorobiphenyls formed from PCBs by plants.

Capacity of enzymes of the biphenyl/chlorobiphenyl pathway, especially biphenyl dioxygenase (BPDO) of two polychlorinated biphenyls (PCB) degrading bacteria, Burkholderia sp. LB400 and Comamonas testosteroni B-356, to metabolize ortho-substituted hydroxybiphenyls was tested.,These compounds found among plant products of PCB metabolism, are carrying chlorine atoms on the hydroxyl-substituted ring. The abilities of His-tagged purified LB400 and B-356 BPDOs to catalyze the oxygenation of 2-hydroxy-3-chlorobiphenyl, 2-hydroxy-5-chlorobiphenyl and 2-hydroxy-3,5-dichlorobiphenyl were compared. Both enzyme preparations catalyzed the hydroxylation of the three chloro-hydroxybiphenyls on the non-substituted ring. Neither LB400 BPDO nor B-356 BPDO oxygenated the substituted ring of the ortho-hydroxylated biphenyl. The fact that metabolites generated by both enzymes were identical for all three hydroxychlorobiphenyls tested; exclude any other mode of attack of these compounds by LB400 BPDOs than the ortho-meta oxygenation.

Diurnal variation in p42/44 mitogen-activated protein kinase in the rat pineal gland.

In this study, we investigated whether there was a diurnal difference in mitogen-activated protein kinase (p42/44(MAPK)) phosphorylation in the rat pineal gland. Under a lighting regimen with 12h of darkness, there was a two- to four-fold increase in phosphorylated levels of MAPK kinase 1/2 (MEK1/2) and p42/44(MAPK) 2h after onset of darkness, an increase that was sustained for 8h. The increases in phosphorylated levels of MEK1/2 and p42/44(MAPK) occurred without increases in MEK1/2 and p42/44(MAPK) proteins. When rats were treated with propranolol 1h before onset of darkness or subjected to continuous light exposure during the dark phase, the nocturnal increase in MEK1/2 and p42/44(MAPK) phosphorylation was reduced. Acute light exposure during darkness caused a decline in MEK1/2 and p42/44(MAPK) phosphorylation within 30 min of light exposure. These results indicate the presence of a diurnal difference in MEK1/2 and p42/44(MAPK) phosphorylation in the rat pineal gland that is under adrenergic control.

Effect of oxygen levels in villous trophoblast apoptosis.

In pregnancies complicated by intrauterine growth restriction (IUGR), the villous trophoblast shows increased apoptosis and immature cytotrophoblasts (CT) may be exposed to both higher or lower oxygen levels than normal placentae. We propose that villous CT undergo higher frequencies of apoptosis at extreme oxygen tensions. The apoptosis of CT isolated from normal term placentae was examined before culture and after 24 h of culture at different oxygen tensions with or without TNFalpha. The apoptosis frequencies of cells cultured for 24 h at O2 levels of approximately 15 mm and approximately 38 mm Hg were similar to the frequency before culture. Both constitutive and TNFalpha-induced apoptosis and cell loss were highest at low (<10 mm Hg) and high ( approximately 140 mm Hg) oxygen tensions. Further, the ratios of induced to constitutive apoptosis, constant from approximately 15 mm to approximately 140 mm Hg, indicate induced apoptosis to be rather insensitive to changes in oxygen levels. These results show that primary villous trophoblasts from normal placentae undergo minimal apoptosis unless subjected to extreme oxygen tensions <15 mm or 140 mm Hg. The results indicate that normal villous trophoblasts are remarkably resistant to hypoxia-induced apoptosis.

Regulation of 90-kilodalton ribosomal S6 kinase phosphorylation in the rat pineal gland.

In this study we investigated diurnal changes in the activation state of the 90-kDa ribosomal S6 kinase (p90RSK) in the rat pineal gland. In animals housed under a lighting regimen with 12 h of light, we found an increase in phosphorylated p90RSK during the dark phase, and this increase was abolished by treatment with propranolol or continuous exposure to light. To determine the intracellular mechanism involved, rat pinealocytes were treated with norepinephrine. Norepinephrine caused a parallel increase in phosphorylated p42/44 MAPK (p42/44(MAPK)) and p90RSK that was reduced by prazosin or propranolol, indicating involvement of both alpha(1)- and beta-adrenergic receptors. Treatment with dibutyryl cGMP, 4beta-phorbol 12-myristate 13-acetate, or ionomycin mimicked norepinephrine-stimulated p90RSK phosphorylation, whereas dibutyryl cAMP caused a decrease in p90RSK phosphorylation. Inhibition of p42/44(MAPK) activation by UO126 was effective in reducing norepinephrine-stimulated p90RSK phosphorylation. Moreover, UO126 had an inhibitory effect on norepinephrine-stimulated arylalkyl-N-acetyltransferase activity. These results indicate that the adrenergically regulated nocturnal increase in p90RSK phosphorylation is mainly mediated through a cGMP-->p42/44(MAPK)-dependent mechanism.

Application of selectively acylated glycosides for the alpha-galactosidase-catalyzed synthesis of disaccharides.

4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the alpha-galactosidase from T. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.

The effect of oxygen tension on intracellular survival signalling in primary villous trophoblasts.

Villous trophoblasts undergo increased apoptosis and experience a wider gradient of oxygen tensions (pO(2)) in pregnancies complicated by intrauterine growth restriction. We hypothesize that pO(2)affects trophoblast apoptosis by altering survival signalling through the phosphatidylinositol-3 (PI-3)-kinase and mitogen activated protein kinase (MAPK) pathways. Cytotrophoblasts were cultured at pO(2)from <10 to approximately 140 mmHg with Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF) at concentrations of 0.1 to 10 ng/ml for 1 to 12 h, then assessed for apoptosis (TUNEL) and specific protein expressions (Western blot analysis). Spontaneous apoptosis was highest at <10 mmHg and lowest approximately 15 mmHg. Only EGF activated either signalling pathway at any pO(2). Inhibition of both pathways was required to inhibit EGF-stimulated survival. Maximal EGF activation of either pathway was insensitive to pO(2). At lower oxygen tensions, MAPK phosphorylation was maximal at 1 ng/ml EGF compared with 10 ng/ml for the PI-3-kinase path. The EGF receptor was spontaneously phosphorylated with increasing culture times at lower oxygen levels, an effect reflected down-stream by PI-3-kinase and Akt phosphorylation. We conclude that strong survival signalling in trophoblasts requires both PI-3- and MAP-kinase pathways, is rather insensitive to pO(2)changes and is spontaneously activated with increasing hypoxic exposure.

Age-related extracellular to total body water volume ratio (Ecv/TBW)--can it be used for "dry weight" determination in dialysis patients? Application of multifrequency bioimpedance measurement.

The article suggests a novel method for quantitative determination of optimal dry weight in dialysis patient based on their extracellular volume (ECV) to total body water (TBW) ratio and its relation to age. Values of ECV and TBW are evaluated by means of whole body multifrequency bioimpedometry. In an effort to find a suitable marker of hydration status in an individual from bioimpedance data, significant correlation has been found between ECV/TBW ratio and age in health. Assuming that all excess fluid in dialysis patients is stored exclusively in ECV and that distribution of their TBW at the state of optimal dry weight corresponds to that of a healthy person of the same age, the pre-dialysis ECV/TBW could be used for quantitative determination of optimal dry weight and/or of the ultrafiltration to reach this weight. Practical bioimpedance measurement of ECV/TBW in a group of dialysis patients both pre- and post-dialysis confirmed both above assumptions, i.e. nearly exclusively extracellular origin of ultrafiltration as well as normalisation of the ECV/TBW ratio towards the end of dialysis. Supporting evidence of increasing ECV/TBW value with age was also found in literature. Although the suggested method needs detailed analysis of possible disturbing factors (ethnic "specificity" of the reference ECV/TBW vs. age characteristics in health, possible difference in "biological" and "physical" age of dialysis patient and others), the article is published at this early stage to enable wider testing of the proposed novel method by different investigators.

Rhythms of the pineal N-acetyltransferase mRNA and melatonin concentrations during embryonic and post-embryonic development in chicken.

Development of a daily rhythmicity in transcription of a gene encoding a rate-limiting enzyme of melatonin biosynthesis, the arylalkylamine-N-acetyltransferase (AA-NAT) was studied by northern blot analysis in pineal glands of 16 and 19-day-old embryos and 1, 4, 8, 11, and 14-day-old chicks. In a parallel experiment, melatonin content in pineal glands and plasma was measured. A significant rhythm of AA-NAT expression was found at embryonic day (ED) 16, the earliest day assayed in this experiment. Expression was low during the daytime and a clear signal was found in the middle of the darktime. The intensity of the signal was increasing during the ontogeny. The nocturnal pineal melatonin concentrations were increasing over the studied period (from ED 19 until post-embryonic day 21). Midnight plasma melatonin concentrations increased from ED19 to PD 3 and oscillated around this value afterwards. Data show that rhythmic expression of AA-NAT mRNA starts very early in development of chicken and plays a major role in melatonin rhythm generation during embryonic development.

Potentiation of cyclic AMP and cyclic GMP accumulation by p38 mitogen-activated protein kinase (p38MAPK) inhibitors in rat pinealocytes.

The effects of p38 mitogen-activated protein kinase (p38MAPK) inhibitors on the adrenergic-stimulated cyclic nucleotide production in rat pinealocytes were investigated. Treatment with SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)IH-imidazole] and SB203580 [4-(4-fluoropheny)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole] (1-100 microM), two pyridinyl imidazole compounds that inhibit p38MAPK, as well as SB202474 [4-(ethyl)-2-(4-methoxyphenyl)-5-(4-pyridyl)IH-imidazole], an inactive analog, was effective in potentiating norepinephrine- and isoproterenol-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation in a concentration-dependent manner. All three compounds caused a greater increase in the cGMP than the cAMP response, with SB202474 being substantially more potent than the two active analogs. At 100 microM, SB202474 potentiated the isoproterenol-stimulated cAMP and cGMP accumulation by 65 and 500%, respectively. Pharmacological studies indicated that the potentiating effect of SB202474 was independent of protein kinase C activation, intracellular calcium elevation, or serine/threonine phosphatase inhibition, three pathways known to potentiate the beta-adrenergic-stimulated cyclic nucleotide responses in rat pinealocytes. In contrast, the potentiating effect of SB202474 was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine. At 100 microM, all three compounds inhibited cAMP- and cGMP-phosphodiesterase activities by 50 and 80%, respectively. These results suggest that the commonly used p38MAPK inhibitors can modulate cyclic nucleotide responses through phosphodiesterase inhibition, a mechanism that appears to be independent of p38MAPK inhibition.

p38MAPK inhibition enhances basal and norepinephrine-stimulated p42/44MAPK phosphorylation in rat pinealocytes.

Interaction between p38MAPK and p42/44MAPK in rat pinealocytes was examined by determining the effects of p38MAPK inhibitors on the phosphorylation of p42/44MAPK using Western blot analysis. Treatment with SB202190, a specific inhibitor of p38MAPK, increased p42/44MAPK phosphorylation in a concentration-dependent manner. SB202190 also enhanced the magnitude and the duration of norepinephrine-activated p42/44MAPK phosphorylation. The effect of SB202190 on p42/44MAPK phosphorylation was abolished by PD98059 or UO126, inhibitors of MEK, suggesting that SB202190 is acting upstream of MEK in activating p42/44MAPK. The SB202190-induced phosphorylation of p42/44MAPK was not blocked by inhibitors of cGMP-dependent kinase (KT5823), protein kinase C (calphostin C) or Ca2+/calmodulin dependent kinase (KN93) suggesting that these pathways may not be involved in the effect of SB202190. SB202190 further increased p42/44MAPK phosphorylation that was stimulated by 8-bromo-cGMP, 4beta phorbol 12-myristate 13-acetate, or ionomycin. In contrast, inhibition of p42/44MAPK phosphorylation by dibutyryl-cAMP persisted when p42/44MAPK phosphorylation was increased by SB202190. Furthermore, inhibition of p42/44MAPK phosphorylation had no effect on p38MAPK activation. These results suggest that inhibition of p38MAPK causes activation of p42/44MAPK and acts synergistically with norepinephrine in the regulation of p42/44MAPK activation in rat pinealocytes.

Lipase-mediated hydrolysis of blackcurrant oil.

Four commercially available lipases, both free and immobilized, were tested for their ability to catalyze hydrolysis of blackcurrant (Ribes nigrum) oil using two different approaches. The lipase from Mucor miehei was studied free and immobilized in two different ways. The former series of enzymic reactions were performed in tap water at 40 degrees C, but the latter series of enzymic processes were carried out in mixtures of isooctane and phosphate buffer (in a typical 2/1 ratio of the components) at 30 degrees C. These conditions were optimized to increase and/or to maximize the yields of the products, which were priority targets in this study. A rate of hydrolysis and a selective preference of the hydrolytic enzymes towards fatty acids, with a special focus on enrichment of alpha-linolenic acid and/or gamma-linolenic acid, were studied. Higher rates of hydrolysis of the blackcurrant oil in the former series of reactions were observed with the immobilized lipase from Pseudomonas cepacia used as biocatalyst. In the latter approach, the most favorable results of the rate of hydrolysis of the target blackcurrant oil were achieved with the immobilized lipase from Mucor miehei employed as biocatalyst. Only three lipases, selected from a series of lipases tested during this investigation, displayed specificity towards alpha-linolenic acid and gamma-linolenic acid, i.e. the immobilized lipase from P. cepacia, lipase from M. miehei and lipase from P. fluorescens.