Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase.

Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/- mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/- mice had significantly increased levels of Il-1ß and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 µM of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1ß, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

Rapid determination of actinides and (90)Sr in river water.

Nuclear accidents occurred in latest years highlighted the difficulty to achieve, in a short time, the quantification of alpha and beta emitters. Indeed, most of the existing methods, though displaying excellent performances, can be very long, taking up to several weeks for some radioisotopes, such as (90)Sr. This study focuses on alpha and beta radioisotopes which could be accidentally released from nuclear installations and which could be measured by inductively coupled plasma mass spectrometer (ICP-MS). Indeed, a new and rapid separation method was developed for (234,235,236,238)U, (230,232)Th, (239,240)Pu, (237)Np, (241)Am and (90)Sr. The main objective was to minimize the duration of the separation protocol by the development of a unique radiochemical procedure with elution media compatible with ICP-MS measurements. Excellent performances were obtained with spiked river water samples. These performances are characterized by total yields exceeding 80% for all monitored radionuclides, as well as good reproducibility (RSD≤10%, n=12). The proposed radiochemical separation (including counting time) required less than 7h for a batch of 8 samples.

Mannose-6-phosphate/insulin-like growth factor-II receptor expression levels during the progression from normal human mammary tissue to invasive breast carcinomas.

The putative role of mannose-6-phosphate/insulin-like growth factor-II receptor (M6P/IGFII-R) as a tumour suppressor and its value as a prognostic marker of breast cancer was studied in 42 benign breast diseases (BBD), 61 in situ carcinomas (CIS) and 133 invasive carcinomas. The receptor was quantified by immunohistochemistry with a computerised image analyser, using specific polyclonal IGY antibodies. The M6P/IGFII-R level varied markedly according to the different patient samples, but median values and distributions were similar in lesions and normal adjacent glands. However, the receptor level was significantly increased in high-grade ductal carcinomas in situ (DCIS) and decreased in invasive carcinomas relative to adjacent normal tissue. The M6P/IGFII-R protein concentration in invasive breast carcinomas was mostly independent of prognostic parameters: tumour size, histological grade, lymph node (N) invasiveness and oestrogen receptor alpha (ERalpha) status. The only positive correlation was with cathepsin D, the progesterone receptor (PgR) and with patients aged >60 years. These results do not support the hypothesis of a frequent and early inactivation of the M6P/IGFII-R gene in breast cancer. Clinical follow-up of patients might reveal a prognostic value for one of the cathepsin receptors.

Polar lipid and fatty acid distribution in carotenolipoprotein complexes extracted from sea buckthorn fruits.

Sea buckthorn (Hippophae rhamnoides L., fam. Elaeagnaceae) fruits are rich in pigments and lipoproteins located in membranes and the fleshy mesocarp. In spite of many reports concerning the neutral lipids in the mesocarp, no data about the polar lipids and their fatty acid composition are available even though they play important structural and physiological roles in cell membranes and may offer interesting applications as emulsifiers and nutrients in cosmetic preparations. Carotenolipoprotein complexes are located particularly in fruit membranes where polar lipids may function as bridge compounds between the polar (protein) and non-polar (carotenoid) moieties. The fatty acid compositions of total and individual polar lipids separated from carotenolipoprotein complexes were determined by HPTLC and GC. The polar lipids included 61% phospholipids and 39% galactolipids, which contained mainly 16:0, 16:1 (9c), 18:1 (9c), 18:1 (11c) and 18:2 (9c, 12c) fatty acids. Almost all polar lipids showed high ratios of 16:0/16:1 (11c) and 18:1 (9c)/18:1 (11c), and higher quantities of 18 carbon unsaturated fatty acids than of the saturated analogue. Galactolipids proved to be richest in 18:1 (9c) and 18:3 (9c, 12c, 15c) fatty acids, while phospholipids contained higher concentrations of 16:0 and 18:1 (9c).

Dissociated overexpression of cathepsin D and estrogen receptor alpha in preinvasive mammary tumors.

The role of estrogen as a promoter agent of sporadic breast cancer has been considered by assaying, in benign breast disease (BBD) and in situ carcinomas (CIS), 2 markers, the estrogen receptor alpha (ERalpha) and cathepsin D (cath-D) involved in estrogen action on mammary tissue. ERalpha and cath-D were assayed by quantitative immunohistochemistry using an image analyzer in 170 lesions of varying histological risk (94 BBD and 76 CIS), and in "normal" glands close to these lesions. The ERalpha level increased significantly in proliferative BBD with atypia (P < .001), in non-high-grade CIS (P < .001), and in adjacent "normal" glands. ERalpha level was decreased in high-grade ductal CIS (DCIS) and also in adjacent "normal" glands. Cath-D level increased in ductal proliferative BBD (P < or = .01) and in high-grade DCIS (P < or = .003), but not in the other lesions. After menopause, ERalpha level was increased (P = .012) but not cath-D level. According to Mac Neman test, the high-grade DCIS were predominantly ERalpha negative and cath-D positive (P = .0017), and the other CIS were predominantly ERalpha positive and cath-D negative (P = .0002). The 2 markers are overexpressed early in premalignant lesions, but independently. This dissociation suggests a branched model of mammary carcinogenesis involving 1 estrogen-independent pathway with high cath-D and low ERalpha levels (including high-grade DCIS) and 1 estrogen-dependent pathway, with high ERalpha level (including proliferative BBD with atypia and low-grade DCIS). We propose that ERalpha-negative breast cancers may develop directly from high-grade DCIS and that ERalpha assay in preinvasive lesions should be considered in prevention trials with antiestrogens.

In vitro yeast (Saccharomyces cerevisiae) presqualene and squalene synthesis related to substrate and cofactor availability.

Squalene synthase catalyses the synthesis of squalene from trans-farnesyl diphosphate in 2 separate steps requiring NAD(P)H. The kinetics of this enzyme in different fractions extracted from a wild-type Saccharomyces cerevisiae were studied. Although this protein is known to be a membrane-bound enzyme, we have found a cytosolic squalene synthase activity besides the microsomal enzyme. A spectrophotometric enzyme assay, not involving isotopic labelling, was established. The relative synthesis of presqualene and squalene was evaluated by using different substrate and cofactor concentrations during the incubation. The involvement of a single catalytic site promoting the 2 reactions of squalene synthesis is suggested.

Evidence for a geranyl-diphosphate synthase located within the plastids of Vitis vinifera L. cultivated in vitro.

Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit geranyl-diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an isopentenyl diphosphate/dimethylallyl diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.

Solubilization and partial purification of squalene synthase from daffodil microsomal membranes.

A squalene synthase was solubilized from daffodil (Narcissus pseudonarcissus L.) microsomes with CHAPS, a zwitterionic non-denaturating detergent. By successive chromatography on DEAE Sephacel and APP Sepharose a fraction enriched in this enzyme (21-fold) was prepared.

Some characteristics of terpenoid biosynthesis by leucoplasts of Citrofortunella mitis.

The capacity of leucoplasts to synthesize monoterpene hydrocarbons has been further investigated by studying the synthesis of higher-terpene homologues. Isolated leucoplasts were unable to esterify chlorophyllides into chlorophylls and to synthesize carotenoids. In addition, the later steps of α-tocopherol and phylloquinone synthesis were not performed. In spite of the presence of a prenyltransferase activity leading to the synthesis of alcohol terpenes higher than gerniol (farnesol, geranyl-geraniol), these higher terpenes were not further metabolized in vitro by leucoplasts.

Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu : Steps and conditions of biosynthesis.

Leucoplasts of immature calamondin and satsuma fruits were incubated with [1-(14)C] isopentenyl pyrophosphate under various conditions. Optimal incorporation of the tracer into geranyl pyrophosphate and monoterpene hydrocarbons occurred in the presence of exogenous dimethylallyl pyrophosphate and Mn(2+) which was more effective than Mg(2+). The dependence of dimethylallyl pyrophosphate showed that about 10 moles were required for 1 mole of isopentenyl pyrophosphate for the best recovery in monoterpene hydrocarbon biosynthesis. A time-course incorporation of isopentenyl pyrophosphate revealed that the C10 hydrocarbon elaboration was dependent on the geranyl pyrophosphate production and at no time neryl pyrophosphate was synthesized by leucoplasts. The amount of labelled farnesyl pyrophosphate was rather low whatever the conditions used in the experiments and sesquiterpene hydrocarbon biosynthesis was never observed.

Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis.

A plastid vesicle preparation isolated from exocarpium of young Citrofortunella mitis (calamondin) fruits was able to synthesise monoterpene hydrocarbons when incubated with isopentenyl pyrophosphate. The electron-microscope comparison between this organelle fraction and the various plastid classes present in the peel tissues has shown the structural identity between these plastid vesicles and the leucoplasts of the epithelial cells lining the secretory pockets. The monoterpene biosynthesis required the presence of dimethylallyl pyrophosphate, Mn(2+) or Mg(2+) and was increased by addition of 2-mercaptoethanol. Evidence is provided that the leucoplast vesicles act as a complete system in which occur all the successive steps involved in monoterpene hydrocarbon elaboration from isopentenyl pyrophosphate.