Characterization and differentiation of pollen lipidomes and proteomes from different intrafloral stamens in heterantherous Senna bicapsularis.

Numerous compounds in pollen can affect the foraging decision-making of bees. Clarification of phytochemical components and identification of key substances for bee foraging preference in pollen are essential steps for apiculture and developing a conservation strategy. Senna bicapsularis, a heterantherous plant that possesses three different stamen types in the same flower, among which bees forage selectively, provides us with an ideal research model for identification of potential substances of bee foraging preference. The lipid and protein compositions of pollen from the anthers of different stamens of S. bicapsularis were investigated and compared. The polyunsaturated fatty acids (PUFAs) and monounsaturated FAs (MUFAs) were highest among lipid molecules in pollen from short (S) stamens than from long (L) and medium (M) stamens. This result is consistent with the FA content measurement, showing the highest FAs and UFAs content in S pollen, especially α-linolenic acid. We inferred that α-linolenic acid might be one of the key substances for bee foraging preference in pollen. Moreover, proteomic analysis showed that several differentially expressed proteins involved in lipid biosynthesis were highly accumulated in S pollen, such as choline kinase 2, 3-oxoacyl-ACP synthase-like protein and choline/ethanolamine phosphotransferase 1, in line with the highest FA content of S pollen. Additionally, DEPs involved in 'starch and sucrose metabolism', 'phenylpropanoid biosynthesis' and 'cyanoamino acid metabolism' were more represented in S compared with L and M pollen. The study suggests that differences in proteomic and lipidomic profiling among the three different stamen types might affect foraging decision-making of bumblebees.

Exploiting altered patterns of choline kinase-alpha expression on human prostate tissue to prognosticate prostate cancer.

AIMS: Malignant transformation results in overexpression of choline-kinase (CHK) and altered choline metabolism, which is potentially detectable by immunohistochemistry (IHC). We investigated the utility of CHK-alpha (CHKA) IHC as a complement to current diagnostic investigation of prostate cancer by analysing expression patterns in normal (no evidence of malignancy) and malignant human prostate tissue samples. METHODS: As an initial validation, paraffin-embedded prostatectomy specimen blocks with both normal and malignant prostate tissue were analysed for CHKA protein and mRNA expression by western blot and quantitative reverse transcriptase PCR (qRT-PCR), respectively. Subsequently, 100 paraffin-embedded malignant prostate tumour and 25 normal prostate cores were stained for both Ki67 (labelling-index: LI) and CHKA expression. RESULTS: The validity of CHKA-antibody was verified using CHKA-transfected cells and siRNA knockdown. Immunoblotting of tissues showed good resolution of CHKA protein in malignant prostate, verifying use of the antibody for IHC. There was minimal qRT-PCR detectable CHKA mRNA in normal tissue, and conversely high expression in malignant prostate tissues. IHC of normal prostate cores showed mild (intensity) CHKA expression in only 28% (7/25) of samples with no Ki67 expression. In contrast, CHKA was expressed in all malignant prostate cores along with characteristically low proliferation (median 2% Ki67-LI; range 1-17%). Stratification of survival according to CHK intensity showed a trend towards lower progression-free survival with CHK score of 3. CONCLUSIONS: Increased expression of CHKA, detectable by IHC, is seen in malignant lesions. This relatively simple cost-effective technique (IHC) could complement current diagnostic procedures for prostate cancer and, therefore, warrants further investigation.

A new family of choline kinase inhibitors with antiproliferative and antitumor activity derived from natural products

Background. Choline kinase alpha (ChoKα) is a critical enzyme in the synthesis of phosphatidylcholine, a major structural component of eukaryotic cell membranes. ChoKα is overexpressed in a large variety of tumor cells and has been proposed as a target for personalized medicine, both in cancer therapy and rheumatoid arthritis. Materials and methods. Triterpene quinone methides (TPQ) bioactive compounds isolated from plants of the Celastraceae family and a set of their semisynthetic derivatives were tested against the recombinant human ChoKα. Those found active as potent enzymatic inhibitors were tested in vitro for antiproliferative activity against HT29 colorectal adenocarcinoma cells, and one of the active compounds was tested for in vivo antitumoral activity in mice xenographs of HT29 cells. Results. Among 59 natural and semisynthetic TPQs tested in an ex vivo system, 14 were highly active as inhibitors of the enzyme ChoKα with IC50 <10 μM. Nine of these were potent antiproliferative agents (IC50 <10 μM) against tumor cells. At least one compound was identified as a new antitumoral drug based on its in vivo activity against xenographs of human HT-29 colon adenocarcinoma cells. Conclusions. The identification of a new family of natural and semisynthetic compounds with potent inhibitory activity against ChoKα and both in vitro antiproliferative and in vivo antitumoral activity supports further research on these inhibitors as potential anticancer agents. Their likely role as antiproliferative drugs deserves further studies in models of rheumatoid arthritis (AU)

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Significantly increased monounsaturated lipids relative to polyunsaturated lipids in six types of cancer microenvironment are observed by mass spectrometry imaging.

Six different types of cancer (i.e., breast, lung, colorectal, esophageal, gastric, and thyroid cancer) have high rates of incidence or mortality worldwide. It has been shown that activation of de novo lipogenesis is an early and common event in the cancer microenvironment. In this study, we performed lipid imaging and profiling for 134 tissue samples from six different types of cancer using matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry, with 2,5-dihydroxybenzoic acid and 1,8-bis(dimethyl-amino)naphthalene as matrices in the positive and negative ion modes, respectively. Multivariate statistical analysis coupled with lipid distribution images revealed that significantly increased levels of monounsaturated fatty acids and monounsaturated phosphatidylcholines relative to polyunsaturated fatty acids and polyunsaturated phosphatidylcholines were observed in the cancer microenvironment compared with the adjacent normal tissue. The immunohistochemical assay indicated that fatty acid synthase, stearoyl-CoA desaturase-1, and choline kinase α were up-regulated in the cancer microenvironment compared with the adjacent normal tissue. Our findings suggest that de novo lipogenesis was activated in six types of cancer to promote a biosynthesis of lipids with monounsaturated acyl chains and to suppress a biosynthesis of polyunsaturated lipids in the cancer microenvironment.

Highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms.

BACKGROUND: Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and ß). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKß and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples. CONCLUSION/SIGNIFICANCE: Apart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.

Increased choline kinase activity in human breast carcinomas: clinical evidence for a potential novel antitumor strategy.

Choline kinase (ChoK) and its product, phosphocholine (PCho), have been implicated in human carcinogenesis. Inhibition of this enzyme has been shown to be an efficient antitumor strategy in vivo. The aim of this study was to assess the relationship between the regulation of ChoK and clinical features in patients with breast cancer. To that end, normal and tumoral tissues from 53 patients with breast carcinomas were analysed for ChoK activity and expression, and compared to some clinical parameters. We report a relevant increase in ChoK activity in 38.5% of the tumoral tissues analysed when compared to the normal levels in healthy tissues. Furthermore, some clinical features were found significant versus ChoK status. First, an association of choline enzymatic activity with histological tumor grade was observed (P<0.001). In addition, increased ChoK activity was also associated with ER-negative breast carcinomas (P=0.037). A significant association between ChoK overexpression and both high histologic tumor grade (P=0.017) and ER-negative tumors (P=0.003) was found. Finally, ChoK overexpression was found in 17% of the samples and all corresponded with those that display the highest increase in ChoK activity (P<0.001). Here we provide evidence that ChoK may be related to the development of human breast cancer, suggesting that this finding may constitute the basis for the development of a novel antitumoral strategy for these patients.

Lidocaine inhibits choline uptake and phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells.

The effect of lidocaine on [3H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte-like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose-dependent manner; at 3.2 mM it resulted in a drastic reduction, by as much as 65 per cent (n = 10; p < 0.0005) or 55 per cent (n = 10; p < 0.0006) in a 3- or 6-h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose-dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3- or 6-h incubation, respectively. Analysis of choline-containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3.2 mM lidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [3H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse-chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until 3H-incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP-choline pathway for PC biosynthesis. After a 3-h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline uptake profoundly in U937 cells.

Gestational age dependence of 11 beta-hydroxysteroid dehydrogenase and its relationship to the enzymes of phosphatidylcholine synthesis in lung and liver of fetal rat.

Increase in fetal surfactant synthesis and lung maturity is caused by the glucocorticoidal induction of enzymes required for phosphatidylcholine (PC) synthesis towards the end of gestation. The regulation of gestational age-dependent induction of PC synthesis by glucocorticoids is still unclear. Since 11-beta-hydroxysteroid dehydrogenase (11 beta-HSD) activity and its metabolising capacity for glucocorticoids have been suggested to play a central role in this regulation, we measured the gestational age-dependent changes in 11 beta-HSD and PC synthesizing enzymes in lung and liver of fetal rat. The activity of cholinephosphate cytidyltransferase (CCT; key enzyme in PC synthesis), choline phosphotransferase (CPT) and lysolecithin acyltransferase (LAT) were found to increase gradually in the lung towards the end of gestation, reached peak values at term followed by a decrease of activity reaching finally adult levels. Only CK activity exhibited constant levels until term followed by a slight increase after the birth. In comparison with the lung, the liver enzymes followed a similar pattern, but at a higher rate of activity except for CCT which was higher in the lung. The activity of 11 beta-HSD in fetal lung microsomes was detectable from day 20 and increased towards the end of gestation in the lung and liver of the rat. Oxidase activity was always found to exceed the reductase activity. The activity of 11 beta-HSD continued to increase after delivery and reached peak levels in adult animals in both organs. In order to test the hypothesis, whether 11 beta-HSD activity and PC synthesis are induced by increasing endogenous glucocorticoidal levels, we examined on day 19 of gestation the effect of dexamethasone (DEXA) on enzymatic activities (11 beta-HSD, CCT) and on [14C]choline incorporation in phosphatidylcholine in fetal lung organoid cultures. Additionally, changes in CCT activity in fetal lungs after maternal administration of DEXA were measured. DEXA accelerated 11 beta-HSD and CCT activities as well as [14C]choline incorporation. We conclude, that endogenous glucocorticoids induce PC synthesis as well as 11 beta-HSD activity in lung and liver of the fetal rat. Fetal PC synthesis is not altered by increasing 11 beta-HSD levels, because the increase of free serum corticosterone levels apparently exceeds the metabolising capacity of 11 beta-HSD towards term.

Effects of fasting on phosphatidylcholine biosynthesis in hamster liver: regulation of cholinephosphotransferase activity by endogenous argininosuccinate.

The control of phosphatidylcholine biosynthesis in the hamster liver was examined. Livers of hamsters fasted for 24 and 48 h were perfused with labelled choline. Under both fasting conditions, the incorporation of labelled choline into phosphatidylcholine was reduced. After 48 h of fasting, the 52% reduction in phosphatidylcholine biosynthesis was caused by changes in several factors including a diminishing rate of choline uptake and severe reductions in the pool sizes of ATP and CTP (to 33-37% control values) which resulted in a decrease in the pools of choline-containing metabolites. The activation of cytidylyltransferase after 48 h of fasting might be regarded as a compensatory mechanism for the maintenance of phosphatidylcholine biosynthesis. After 24 h of fasting, a 25% reduction in phosphatidylcholine biosynthesis was observed. The ATP and CTP levels were decreased but the reduction was not severe enough to affect the choline uptake or the labelling of the phosphocholine fraction. The activities of the cytidylyltransferase remained unchanged but an accumulation of labelled CDP-choline was detected. Although choline-phosphotransferase activity was not changed in the microsomes, the enzyme activity was attenuated in the postmitochondrial fraction. Further analysis revealed that cholinephosphotransferase in the liver was inhibited by an endogenous inhibitor in the cytosol which was later identified as argininosuccinate. The level of argininosuccinate was elevated during fasting and the change quantitatively accounted for the attenuation of cholinephosphotransferase activity. The inhibition of choline-phosphotransferase by argininosuccinate, coupled with a substantial decrease in the diacylglycerol level, would provide the hamster liver with an immediate mechanism for the transient modulation of phosphatidylcholine biosynthesis during short-term fasting.

Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae.

1. The structural gene for cholinephosphate cytidylyltransferase (CCT) was isolated from a Saccharomyces cerevisiae genomic library by means of complementation in a mutant of the yeast defective in the enzyme. The cloned DNA restored both the growth and cholinephosphate cytidylyltransferase activity of the mutant. Whereas the enzyme of the mutant was thermolabile, the enzyme produced by the transformant was indistinguishable in heat stability from that produced by the wild type. 2. Strains carrying a multicopy recombinant plasmid overproduced cholinephosphate cytidylyltransferase. The overproduction of the enzyme brought about an increase in the synthesis of CDPcholine in the transformant, but there was no increase in the overall rate of phosphatidylcholine synthesis. 3. The cloned DNA was subcloned into a 2.5-kb DNA fragment. The nucleotide sequence which contained CCT was determined by the dideoxy chain-termination method. The sequence contained an open reading frame capable of encoding a protein of 424 amino acid residues with a calculated relative molecular mass of 49,379.31. Northern blot analysis showed that this DNA segment is transcribed in yeast cells and the length of the transcript is consistent with the putative translation product. 4. Hydropathy analysis according to Kyte and Doolittle indicated that the primary translation product contains extended hydrophilic stretches in its N- and C-terminal regions. 5. The primary translation product contains a region showing local sequence homology with nucleotidyl-transfer enzymes such as DNA polymerase (Escherichia coli), CDPdiacylglycerol pyrophosphatase (E. coli), 3-deoxy-manno-octulosonate cytidylyltransferase (E. coli) and DNA ligase (T4 phage), suggesting that these five enzymes are evolutionarily related. Statistically significant sequence homology was also noted between the human c-fos gene product and the enzyme.

Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine.

In Plasmodium falciparum-infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 +/- 1.4 nmol phosphoethanolamine/10(7) infected cells per h) was higher than choline kinase specific activity (1.9 +/- 0.2 nmol phosphocholine/10(7) infected cells per h). The Km of choline kinase for choline was 79 +/- 20 microM, and ethanolamine was a weak competitive inhibitor of the reaction (Ki = 92 mM). Ethanolamine kinase had a Km for ethanolamine of 188 +/- 19 microM, and choline was a competitive inhibitor of ethanolamine kinase with a very high Ki of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D-2-amino-1-butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50 degrees C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism.

The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity.

The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline-phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast-pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast-pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase.

Activities of the phosphatidylcholine biosynthetic enzymes in rat liver during development.

The activities of the enzymes of rat hepatic phosphatidylcholine biosynthesis have been measured as a function of development in the rat (term, 23 days). During the last 5 days of gestation, the specific activity of choline kinase was elevated almost fivefold (p less than 0.05). After parturition, choline kinase activity was reduced to adult values by the 5th postnatal day. Over 75% of the total CTP:phosphocholine cytidylyltransferase protein in prenatal liver was detected in the cytosolic fraction. On the day of birth, most of the cytidylyltransferase translocated to the microsomes so that the microsomal specific enzyme activity was 3.3-fold higher (p less than 0.01) and the cytosolic specific enzyme activity (measured in the presence of phospholipid) was 68% lower (p less than 0.001) than the day before parturition. CDPcholine:diacylglycerol cholinephosphotransferase activity (measured in the presence of diacylglycerol) increased 130-fold (p less than 0.001) during the last 5 days of gestation. On the 10th postnatal day, cholinephosphotransferase activity was 1.7-fold higher (p less than 0.001) than immediately after birth, but declined to adult values by the 19th day. Between the 5th day prior to parturition and the 10th postnatal day, phosphatidylethanolamine N-methyltransferase activity steadily increased 16-fold (p less than 0.001). The results are in agreement with the hypothesis that the increase in phosphatidylcholine in rat liver during the perinatal period is due to an increased synthesis of CDPcholine, which is a consequence of the translocation of the cytidylyltransferase from cytosol to the endoplasmic reticulum.

[Rapid method for the determination of choline kinase activity. Choline kinase in marine invertebrates]. / Ekspress-metod opredeleniia aktivnosti kholinkinazy. Kholinkinaza morskikh bespozvonochnykh.

A quick sensitive test is suggested to determine choline kinase (EC 2.7.1.32). The method includes incubation of a substrate with enzyme in microvolume and separation of the formed phosphorylcholine from the initial choline by high-performance thin-layer silica gel chromatography. The procedure of determination is simple, reproducible and takes no more than 30 min. The method reveals high activity of choline kinase in some marine invertebrates. Certain features of the enzyme from the intestine of strongylocentrotus intermedius are described.

Familial respiratory distress syndrome in three consecutive full-term infants. Case reports and documentation of lung enzyme activities.

Familial respiratory distress syndrome in full-term newborn infants is a rare occurrence. Our patient delivered three consecutive full-term infants who developed findings consistent with respiratory distress syndrome. All three died from autopsy-proven hyaline membrane disease. Analysis of the activities of four enzymes that play an important role in the biosynthesis of lecithin (choline kinase, choline phosphotransferase, phospholipase A and lysolecithin acyltransferase) failed to disclose an abnormality in lung samples in our patient with familial respiratory distress syndrome.