Synthesis and in vitro activities of a spacer-containing glycophospholipid ligand of a lipopolysaccharide receptor involved in endotoxin tolerance.

A glycophospholipid consisting in a derivative of N,N'-acylated and bisphosphorylated 2,3-dideoxy-2,3-diamino-D-glucose, bearing a 6-aminocaproyl side chain as spacer arm at carbon 6 (PPDm2-B), has been synthesized and its effect on murine macrophages evaluated. The synthesis started from 2,3-diamino-D-glucose, which was best obtained from glucosamine essentially by known procedures, since attempts to use another known precursor (3-nitroglycoside) led to unexpected results. Selective N-acylation was performed with the hydroxysuccinimide ester of (D)-3-benzyloxymyritic acid followed by esterification of the sole primary hydroxyl function by 6-azidocaproylchloride and phosphorylation of the resulting 1,4-diol by treatment with tetrabenzyl pyrophosphate. Hydrogenation on a Pd on carbon catalyst permitted the isolation of 6-(6-aminohexanoyl)-2,3-dideoxy-2,3-di-[(R)-3-hydroxy-tetradecanamido ] -alpha-D-glucopyranose 1,4-diphosphate (PPDm2-B). In mouse macrophages, PPDm2-B enhanced the lipopolysaccharide (LPS)-dependent secretion of tumor necrosis factor alpha (TNF-alpha), and inhibited the LPS-induced desensitization of these cells. The data suggest that PPDm2-B interacts in a serum-independent way with an LPS receptor different from CD14, and involved in endotoxin tolerance. Binding studies of a fluorescent derivative of PPDm2-B indicated that the expression of this unknown receptor is down-regulated during in vitro culture of the cells. Owing to its spacer arm, PPDm2-B could thus be a promising tool for future studies of this receptor.

Steroidal affinity labels of the estrogen receptor. 3. Estradiol 11 beta-n-alkyl derivatives bearing a terminal electrophilic group: antiestrogenic and cytotoxic properties.

With the aim of developing a new series of steroidal affinity labels of the estrogen receptor, six electrophilic 11 beta-ethyl (C2), 11 beta-butyl (C4), or 11 beta-decyl (C10) derivatives of estradiol bearing an 11 beta-terminal electrophilic functionality, i.e. bromine (C4), (methylsulfonyl)oxy (C2 and C4), bromoacetamido (C2 and C4), and (p-tolylsulfonyl)oxy (C10), were synthesized. The range of their affinity constants for binding the estrogen receptor was 0.4-37% that of estradiol; the order of increasing affinity (i) relative to the 11 beta-alkyl arm was ethyl < butyl and (ii) relative to the electrophilic functionality was bromoacetamido < bromine < (methylsulfonyl)oxy. Regardless of the conditions used, including prolonged exposure of the receptor to various pH levels (7-9) and temperatures (0-25 degrees C), the extent of receptor affinity labeling by the 11 beta-ethyl and 11 beta-butyl compounds, if any, was under 10%. This was in sharp contrast to results obtained using 11 beta-((tosyloxy)decyl)estradiol which labeled from 60% to 90% of the receptor hormone-binding sites with an EC50 of approximately 10 nM. Estrogenic and antiestrogenic activities of the compounds were determined using the MVLN cell line, which was established from the estrogen-responsive mammary tumor MCF-7 cells by stable transfection of a recombinant estrogen-responsive luciferase gene. The two 11 beta-ethyl compounds were mainly estrogenic, whereas the three 11 beta-butyl and the 11 beta-decyl compounds essentially showed antiestrogenic activity. The fact that the chemical reactivities of 11 beta-ethyl and 11 beta-butyl compounds were not compromised by interaction with the estrogen receptor made the synthesized high-affinity compounds potential cytotoxic agents which might be able to exert either (i) a specific action on estrogen-regulated genes or (ii) a more general action in estrogen-target cells. Therefore the ability of the compounds (1) to irreversibly abolish estrogen-dependent expression of the luciferase gene and (2) to affect the proliferation of MVLN cells were determined. All electrophiles were able to irreversibly suppress expression of the luciferase gene; the antiestrogenic electrophiles were more potent than the estrogenic ones but less efficient than 4-hydroxytamoxifen, a classical and chemically inert triphenylethylene antiestrogen. Only the antiestrogenic electrophiles decreased cell proliferation; however, they were less potent than 4-hydroxytamoxifen. In conclusion, the synthesized electrophilic estradiol 11 beta-ethyl and 11 beta-butyl derivatives (i) were not efficient affinity labels of the estrogen receptor and (ii) did not display significant cytotoxicity in estrogen-sensitive mammary tumor cells. However, since these derivatives displayed high affinity for the estrogen receptor, they could be used to prepare potential cytotoxic agents which might be selective for tumors affecting estrogen-target tissues, by coupling them with a toxic moiety.

Involvement of the membrane form of tumour necrosis factor-alpha in lipopolysaccharide-induced priming of mouse peritoneal macrophages for enhanced nitric oxide response to lipopolysaccharide.

We studied the pathways of macrophage response to lipopolysaccharide (LPS). When mouse macrophages pre-exposed to LPS were restimulated with this agent, reduced tumour necrosis factor-alpha (TNF-alpha) responses (desensitization/endotoxin tolerance) were accompanied by increased (priming) nitric oxide (NO) responses. Priming was also inducible with recombinant interferon-beta (IFN-beta). The requirement of TNF-alpha biosynthesis in the LPS-induced priming was also suggested by the observation that both anti-TNF-alpha serum and pentoxifylline inhibited this effect. However, addition of mouse recombinant TNF-alpha (mrTNF-alpha) did not enhance the priming induced by LPS or IFN-beta, and preincubation with mrTNF-alpha alone, or in association with other cytokines produced by macrophages (interleukin-1 beta, interleukin-6, or leukaemia inhibitory factor), did not induce a priming effect. We found however, that pentoxifylline, which blocked the priming, also decreased the level of membrane-bound TNF-alpha. Furthermore, exposure to compound BB-3103 (a metalloproteinase inhibitor that blocks the processing of membrane-bound TNF-alpha yielding to the secreted cytokine) enhanced the priming effect, the expression of membrane TNF-alpha and the specific binding of LPS. These observations suggest that the membrane form of TNF-alpha is involved in the interaction of LPS with a receptor required for LPS-induced priming.

Evaluation of the Dacos 3.0 analyser.

The selective multitest Coulter Dacos 3.0 analyser was evaluated according to the guidelines of the Comisión de Instrumentación de la Sociedad Española de Química Clínica and of the European Committee for Clinical Laboratory Standards.THE EVALUATION WAS PERFORMED IN FOUR STEPS: examination of the analytical units; evaluation of routine working; study of interferences; and assessment of practicability.The evaluation included a photometric study. The inaccuracy is acceptable for 340 nm and 420 nm, and the imprecision at absorbances from 0.05 to 2.00 ranged from 0.06 to 0.28% at 340 nm and from 0.06 to 0.08% at 420 nm. The linearity showed some dispersion at low absorbance for PNP at 420 nm and the drift was negligible.The imprecision of the pipette delivery system, the temperature control system and the washing system were satisfactory.IN ROUTINE WORK CONDITIONS, SEVEN ANALYTICAL METHODS WERE STUDIED: glucose, creatinine, iron, total protein, AST, ALP and calcium. Within-run imprecision ranged, at low concentrations, from 0.9% (CV) for glucose, to 7.6% (CV) for iron; at medium concentrations, from 0.7% (CV) for total protein to 5.2% (CV) to creatinine; and at high concentrations, it ranged from 0.6% (CV) for glucose to 3.9% (CV) for ALP.Between-run imprecision at low concentrations ranged from 1.4% (CV) for glucose to 15.1% (CV) for iron; at medium concentrations it ranged from 1.2% (CV) for protein to 6.7% (CV) for iron; and at high concentrations the range is from l.2for AST to 5.7% (CV) for iron.No contamination was found in the sample carry-over study. Some contamination was found in the reagent carry-over study (total protein due to iron and calcium reagents). Relative inaccuracy is good for all the constituents assayed. Only LDH (high and low levels) and urate (low level) showed weak and negative interference caused by turbidity, and gamma-GT (high level) and amylase, bilirubin and ALP (two levels) showed a negative interference caused by haemolysis.