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PubMed1975

[Preparation of immunoglobulins G,A,M (IgGAM) for therapeutic use. Conditions for enrichment in IgA or in IgM].

Antibodies directed against viruses and bacteria are not equally distributed among the main classes of immunoglobulins, e.g. IgG, IgA and IgM. It has been found that IgM is mostly concerned with certain antibacterial activities (Salmonella, Escherichia coli and Pseudomonas) and IgA with high antibody titers for poliomyelitis virus I whereas antibody activities against many viruses such as influenza and measles virus occur preferentially in the IgG population. Furthermore, isolated immunoglobulin deficiency syndromes are actually well known. In the light of these findings, new concepts of immunotherapy have developed. Massive i.v. IgG-therapy is already widely used in congenital and acquired severe hypogammaglobulinemia. Preparations enriched in IgA and IgM are needed to complete the immunotherapeutical possibilities. Such a fraction called IgGAM has already been prepared in our Institute. Fraction III obtained during large scale fractionation is used as starting material and caprylic acid for the precipitation of most proteins other than the immunoglobulins present in fraction III. The immunoglobulin concentrate is finally obtained by ethanol precipitation of the caprylic acid supernatant. The present study is concerned with various modifications of the initial technique in order to obtain fractions more specially enriched in IgA or in IgM. In some cases the standard IgGAM fraction has been submitted to a further fractionation step, such as adsorption of IgG on DEAE-cellulose or precipitation of certain immunoglobulins achieved by Rivanol or by lowering the salt concentration. In other trials the fractionation procedure starting from fraction III has been modified. Rivanol has been used as a precipitating agent for the subfractionation of fraction III. It is well known that IgG is soluble in the presence of Rivanol. This technique was thus used in order to obtain preparations enriched mainly in IgM and IgA. The precipitate obtained after the addition of Rivanol was dissociated by NaCl and the solution further subfractionated by caprylic acid. In a similar way PEG was associated with the caprylic acid precipitation step. PEG precipitates proteins mainly in function of their molecular weight. However, the enrichment of IgM of the final fraction did not exceed 32% and much IgM was lost under the experimental conditions. It proved easiest to suspend fraction III in distilled water leaving IgM in the precipitate; it is dissolved and the solution submitted to a slightly modified caprylic acid precipitation step. This fraction contains 35-40% IgM, few (2-6%) IgA and about 50% IgG whereas an IgA (35%) enriched fraction is obtained when fraction III is solubilized with acetate at pH 6.2 and then submitted to precipitation by caprylic acid under slightly modified conditions as compared with our standard IgGAM. Thus, simple modifications of the standard procedure allow to prepare fractions enriched more specially in IgM or IgA. Fractions poor or almost devoid of IgG can also be obtained...

Caproates↗
PubMed2003

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) via manipulating the fatty acid beta-oxidation pathway in E. coli.

Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalkanoate synthase gene (phaC(Ac)) and (R)-enoyl-CoA hydratase gene (phaJ(Ac)) from Aeromonas caviae. The expression plasmids were introduced into E. coli JM109, DH5 alpha and XL1-blue, respectively. Compared with the strains harboring only phaC(Ac) and phaJ(Ac), all recombinant E. coli strains harboring yafH, phaC(Ac) and phaJ(Ac) accumulated at least four times more poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Cell dry weights produced by all recombinants containing yafH were also considerably higher than that without yafH. The addition of acrylic acid which serves as inhibitor for beta-oxidation and may lead to more precursor supply for PHA synthesis did not result in improved PHBHHx production compared with that of the overexpression of yafH. It appeared that the overexpression of acyl-CoA dehydrogenase gene (yafH) enhanced the supply of enoyl-CoA which is the substrate of (R)-enoyl-CoA hydratase. With the enhanced precursor supply, the recombinants accumulated more PHBHHx.

3-Hydroxybutyric Acid↗
PubMed2000

Effects of 2-ethylhexanoic acid on the production of reactive oxygen species in human polymorphonuclear leukocytes in vitro.

2-Ethylhexanoic acid (2-EHA), is an industrial chemical and a toxic biotransformation product of the plasticizer di(2-ethylhexyl)phthalate. Its immunological effects are unknown. 2-EHA resembles structurally C18 fatty acids, which are known activators of respiratory burst in human polymorphonuclear leukocytes (PMNL). Therefore, we exposed PMNL to 2-EHA in vitro and measured the production of reactive oxygen species (ROS) and explored the associated cellular mechanisms. 2-EHA (10-2000 microM) inhibited dose-dependently formyl-methionyl-leucyl-phenylalanine (FMLP)-induced respiratory burst in PMNL. Moreover, 2-EHA decreased oxidative burst evoked by the protein kinase C (PKC) activators, phorbol myristate acetate (PMA) and dioctanoyl-s,n-glycerol (DIC(8)). 2-EHA affected neither the levels of free intracellular calcium nor inhibited PKC. The results indicate that 2-EHA inhibits activation of PMNL to produce ROS, i.e. has an immunosuppressive effect in vitro. The site of action in the PKC is after activation of this enzyme.

Caproates↗
PubMed2001

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC-MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1,000 microg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1 +/- 13.5 microg/l for 2-ethylhexanoic acid, 104.8 +/- 80.6 microg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2 +/- 389.5 microg/l for 2-ethyl-3-oxohexanoic acid.

Caproates↗
PubMed2003

Study of emulsion stabilization by graft copolymers using the optical analyzer Turbiscan.

Oil-in-water nanoemulsions were prepared using a series of synthetic graft copolymers with a backbone of dextran (DEX) and a number of side chains of poly-epsilon-caprolactone (PCL). In this paper, we focus on the o/w emulsion stabilizing abilities of these novel PCL-DEX copolymers, using a recently developed optical analyzer (Turbiscan). The main advantage of Turbiscan is to detect the destabilization phenomena in non-diluted emulsion, much earlier than the naked eye's operator, especially in the case of an opaque and concentrated system. This study shows that PCL-DEX copolymers successfully stabilized ethyl acetate-in-water emulsions, even in the absence of additional surfactants, whereas they were not efficient in stabilizing methylene chloride-in-water emulsions which coalesced fast and irreversibly. The ethyl acetate-in-water emulsion stabilizing ability of PCL-DEX seemed to be related to the localization of their blocks with regard to the oil-water interface.

Acetates↗
PubMed1999

Primidone-loaded poly-epsilon-caprolactone nanocapsules: incorporation efficiency and in vitro release profiles.

This paper describes the preparation of primidone-loaded poly-epsilon-caprolactone nanocapsules according to the interfacial deposition technique. The colloidal suspension obtained showed a monomodal size distribution with a mean diameter ranging from 308 to 352 nm. By adjusting the process parameters, the encapsulation efficiency was about 74% with good reproducibility. Primidone release from the nanocapsules was found to be slower as compared to the oily control solution despite an important burst-effect. The release profile was not influenced by the pH of the release medium.

Caproates↗
PubMed1999

Automated solid-phase extraction workstations combined with quantitative bioanalytical LC/MS.

An automated solid-phase extraction workstation was used to develop, characterize and validate an LC/MS/MS method for quantifying a novel lipid-regulating drug in dog plasma. Method development was facilitated by workstation functions that allowed wash solvents of varying organic composition to be mixed and tested automatically. Precision estimates for this approach were within 9.8% relative standard deviation (RSD) across the calibration range. Accuracy for replicate determinations of quality controls was between -7.2 and +6.2% relative error (RE) over 5-1,000 ng/ml(-1). Recoveries were evaluated for a wide variety of wash solvents, elution solvents and sorbents. Optimized recoveries were generally > 95%. A sample throughput benchmark for the method was approximately equal 8 min per sample. Because of parallel sample processing, 100 samples were extracted in less than 120 min. The approach has proven useful for use with LC/MS/MS, using a multiple reaction monitoring (MRM) approach.

Animals↗
PubMed1985

[Inhibition of postoperative muscular proteolysis by sodium alpha-ketoisocaproate: does a dose-effect relation exist?].

The inhibitory effect of branched chain amino acids on muscle protein breakdown has been attributed to the ketoanalogue of leucine, alpha-ketoisocaproic acid. In an attempt to demonstrate a possible dose-response relationship for ketoleucine, the postoperative protein breakdown (using urinary excretion of 3-methylhistidine, creatinine and nitrogen) was evaluated in 29 patients undergoing gynaecological surgery. They were randomly assigned to one of the following groups: group A (ten patients) received 3 g of glucose per kilo and per day, group B (ten patients) received glucose + 100 mg X kg-1 X d-1 of ketoleucine and group C (nine patients) glucose + 200 mg X kg-1 X d-1 of ketoleucine. The study was performed over a period of 72 h. The 3-methylhistidine/creatinine ratio was significantly lower in groups B and C than in group A (respectively 28.47, 28.07 and 32.08). These results confirmed the inhibitory effect of ketoleucine on muscle protein breakdown. However there was no difference between group B and group C. In conclusion, this study did not demonstrate a dose-response relationship for ketoleucine in moderate postoperative catabolism.

Adult↗
PubMed1998

Signal transduction in islet hormone release: interaction of nitric oxide with basal and nutrient-induced hormone responses.

We examined the relation between the islet NO system and islet hormone secretion induced by either the non-glucose nutrient alpha-ketoisocaproic acid (KIC) or, in some experiments, glucose. KIC dose dependently stimulated insulin but inhibited glucagon secretion. In a medium devoid of any nutrient, the NO synthase (NOS)-inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) induced an increase in basal insulin release but a decrease in glucagon release. These effects were evident also in K+-depolarised islets. KIC-induced insulin release was increased by L-NAME. This increase was abolished in K+-depolarised islets. In contrast, glucose- induced insulin release was potentiated by L-NAME after K+ depolarisation. The intracellular NO donor hydroxylamine dose dependently inhibited KIC-stimulated insulin release and reversed KIC-induced suppression of glucagon release. Our data suggest that islet hormone secretion in a medium devoid of nutrients is greatly affected by the islet NO system, whereas KIC-induced secretion is little affected. Glucose-induced insulin release, however, is accompanied by increased NOS activity, the NOS-activating signal being derived from the glycolytic-pentose shunt part of glucose metabolism. The observed NO effects on islet hormone release can proceed independently of membrane-depolarisation events.

Animals↗
PubMed2000

Production, regeneration and biochemical precursors of the major components of the defensive secretion of Eurycotis floridana (Dictyoptera, polyzosteriinae).

The defensive secretion of the cockroach Eurycotis floridana contains three main components, (E)-2-hexenal, (E)-2-hexenol and (E)-2-hexenoic acid, which represented about 98% of the organic phase. The quantity of the aldehyde, alcohol, and acid present in the defensive secretion increased rapidly for 60 days from the imaginal moult. Following artificial discharge, the males were able to regenerate their initial volume of secretion over a 30 day period. To investigate the possible routes of biosynthesis of the three components, E. floridana was injected with 14C-labeled fatty acids and acetate, and the incorporation of 14C into the three components were quantified 1, 6, and 24 h after milking. Our results revealed that oleic, linoleic, linolenic and palmitic acids, which constitute part of the fat body of the insect, were incorporated to the same degree into the three main components, but very slowly compared to acetate. Although it has not been possible to identify the exact route of aldehyde, alcohol and acid biosynthesis, our findings suggests that (E)-2-hexenal, (E)-2-hexenol and (E)-2-hexenoic acid are preferentially biosynthesized de novo from acetate.

Animals↗
PubMed1997

Discovery of OT4003, a novel, potent, and orally active cys-LT1 receptor antagonist.

The present paper describes the structural modifications leading to the discovery of a new series of quinoline-containing cys-LT1 receptor (LTD4 receptor) antagonists. A structural optimization with respect to the in vitro receptor binding, the in vivo brochoconstriction, and the toxicological effect in the form of peroxisomal proliferation was performed in order to achieve the target compound OT4003. OT4003 ((S)-(+)-E-2-(3-(2-(7- chloroquinolin-2-yl)ethenyl)phenylaminomethyl)-phenoxyl++ +-hexanoic acid) was found to be a potent and selective inhibitor of [3H]LTD4 specific binding to guinea pig lung membranes (IC50 2.4 +/- 1.0 nM), and also a potent, orally active, antagonist of LTD4 induced bronchoconstriction in guinea pigs [ED50 0.14 (ED16 0.1-ED84 0.4) mg/kg; 4 h pretreatment]. The enantiomerically pure OT4003 was prepared using a short convergent synthesis, including an enzymatic resolution step.

Administration, Oral↗
PubMed2002

Initial characterization of a type I fatty acid synthase and polyketide synthase multienzyme complex NorS in the biosynthesis of aflatoxin B(1).

The biosynthesis of the potent environmental carcinogen aflatoxin B(1) is initiated by norsolorinic acid synthase (NorS), a complex of an iterative type I polyketide synthase and a specialized yeast-like pair of fatty acid synthases. NorS has been partially purified from Aspergillus parasiticus, has been found to have a mass of approximately 1.4 x 10(6) Da, and carries out the synthesis of norsolorinic acid in the presence of acetylCoA, malonylCoA, and NADPH where hexanoylCoA is not a free intermediate. The N-acetylcysteamine thioester of hexanoic acid can substitute for the catalytic functions of HexA/B to initiate norsolorinic acid synthesis by the complex in the presence of only malonylCoA. An alpha(2)beta(2)gamma(2) stoichiometry is proposed for NorS in keeping with its estimated mass and the observed dimeric or higher-order quarternary structures of PKS and FAS enzymes.

Aflatoxin B1↗
PubMed1999

Lymph node extraction of radiopaque nanoparticulates in the rabbit as measured in vivo with CT.

RATIONALE AND OBJECTIVES: The purpose of this study was to estimate in vivo extraction of lymphographic material in the popliteal node of the rabbit. MATERIALS AND METHODS: Serial quantitative computed tomography (CT) of target tissues in four legs of two rabbits was performed after subcutaneous injection of an improved lymphographic contrast agent. Massage was used as a lymphotrophic intervention. RESULTS: At 15 minutes, the mean change in Hounsfield units measured 815 in the popliteal node, 219 in afferent lymphatic vessels, and 127 in efferent lymphatic vessels. The nodal extraction of nanoparticulates from the lymph was approximately 55%. Nodal massage allowed the amount of nanoparticulate remaining in sinusoidal lymph to be estimated. CONCLUSION: Functional CT performed with timed studies, proper radiopaque materials, and physiologic interventions can depict in vivo lymphatic physiology under minimally invasive conditions.

Animals↗
PubMed2001

Olfactory event-related potentials in patients with brain tumors.

OBJECTIVE: The aim of the study was to determine how odor processing is altered in patients with unilateral supratentorial brain tumors. METHODS: Olfactory event-related potentials (OERPs) were evaluated in 10 patients with unilateral brain tumors of the frontal or temporal lobe in response to linalool and allylcaproate. Both odors were presented monorhinally by a constant-flow olfactometer. In addition, 20 healthy subjects were examined. While sniffing, the subjects were asked to discriminate the two odors. EEG was recorded from 7 electrode positions (Fz, Cz, Pz, F3/4, P3/4). Amplitudes and latencies of 3 peaks (N1, P2, P3) were measured. To control for effects of modality-non-specific alterations on the olfactory components acoustic event-related potentials (AERPs) were registered by use of an oddball paradigm. RESULTS: Patients with right-sided lesions showed distinct deficits in the discrimination task after stimulation of the right and left nostril. In contrast, patients with left-sided lesions only had an attenuation of correct reactions after left-sided stimulation. In the OERPs, patients with right-sided lesions showed P2- and P3-components with decreased amplitudes at parietal electrode positions. These alterations appeared after ipsi- and contralateral stimulation. Patients with left-sided lesions showed a significant effect of the side of stimulation. Their OERP-amplitudes were decreased after left-sided stimulation but not after right-sided stimulation. After right-sided olfactory stimulation a correlation between the olfactory and the acoustic ERP was seen in patients with right-sided lesions. CONCLUSIONS: Olfactory performance of the participating patients was markedly reduced. Patients with right-sided lesions showed bilateral impairment, which would support the importance of the right hemisphere in olfaction. The alteration of the topographic distribution of P2- and P3-amplitudes in patients with right-sided lesions might reflect an impairment of early and late olfactory processing steps.

Acyclic Monoterpenes↗
PubMed2000

Odour-impact compounds of Gorgonzola cheese.

Volatile concentrates were obtained by vacuum distillation from both natural and creamy Gorgonzola cheese and isolated by continuous liquid-liquid extraction. Both were analysed by high resolution gas chromatography (HRGC), HRGC-mass spectrometry and HRGC-olfactometry. A total of 63 components were identified in the neutral extract of the natural type (21 esters, 13 ketones, 14 alcohols, 5 aldehydes, 1 sulphur compound, 7 aromatic compounds and 2 terpenes) and 52 in the creamy type (17 esters, 12 ketones, 10 alcohols, 5 aldehydes, 1 sulphur compound, 5 aromatic compounds and 2 terpenes). Ketones, whose major components were 2-nonanone and 2-heptanone, were the predominant constituents of the neutral fraction. By olfactometric analysis of the neutral extracts, 23 odour-impact compounds were found in the natural and 21 in the creamy Gorgonzola cheese. 1-Octen-3-ol, ethyl hexanoate, 2-nonanone, 2-heptanone, 2-heptanol, ethyl butanoate, 2-nonanol and 4-methylanisole were the key odorants of the natural cheese, whereas 2-heptanone, 2-heptanol, ethyl butanoate, 3-methyl thiopropanal and an unidentified constituent with a fruity odour were characteristic of the creamy Gorgonzola cheese. On the basis of high odour unity values, 2-nonanone, 1-octen-3-ol, 2-heptanol, ethyl hexanoate, methylanisole and 2-heptanone were the most important odorants of natural and creamy Gorgonzola cheese aroma.

Animals↗
PubMed2000

Role of Yersinia intermedia and Pseudomonas putida in the development of a fruity off-flavour in pasteurized milk.

Analysis by gas chromatography-mass spectrometry of pasteurized milk with a fruity (pineapple like) off odour and a sour, rancid and soapy taste indicated the presence of concentrations at microg/ml levels of ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, octanoic acid, decanoic acid and dodecanoic acid. The off-odour and taste were attributed to the presence of these compounds in the milk. Microbiological examination confirmed that the milk was also contaminated with a series of psychrotrophic bacteria including Yersinia intermedia, Pseudomonas putida and Rahnella aquatilis. Growth of isolates of these bacteria in UHT milk at 23 degrees C for 7 d showed that Yer. intermedia produced significant quantities of the C4-C12 alkanoic acids; Ps. putida produced only small quantities of these acids and Rah. aquatilis produced none. In addition, Yer. intermedia and Ps. putida also produced small but significant quantities of the corresponding ethyl esters. In milk inoculated with both Yer. intermedia and Ps. patida, the quantity of ethyl esters produced was greater than that found in cultures containing only one of the isolates. These studies indicated that Yer. intermedia was the principal source of the alkanoic acids in the tainted milk and that the major producer of the corresponding ethyl esters was Ps. patida. This is the first report that Yer. intermedia and Ps. putida can cause an off-odour or taste in dairy products.

Animals↗