Development of a sensitive semi-automatized enzyme immunoassay of leukotriene using acetylcholinesterase-leukotriene C4 as tracer

The obtention of icosanoids tracers of high specific radioactivity (e.g. radioiodinated tracers) has been a prerequisite for the development of radioimmunoassays that would allow the detection of femtomoles amount of these substances from biological medium. However, recent attempts to develope immunoassays using haptens (e.g. prostaglandins or thromboxane B2) labeled with enzymes have turned out to be disappointing because of their poor sensitivity. Using the acetylcholinesterase (AChE) from “electrophorus electricus” as a tracer we have labeled LTC4 after coupling it to the enzyme with 1,5-difluoro-2,4-dinitrobenzene as a bifunctional reagent. The use of 96-well microtiter plates coated with pig anti-rabbit immunoglobulin antibodies (purified by affinity chromatography) has allowed to develop a semiautomatized enzyme immunoassay (EIA). A dispenser was used to add all common reagents (antibody, tracer, enzyme substrate); a washer was used to eliminate the unreacted molecules from the immuno-reactions. After addition of the enzyme substrate (Ellman's reagent), the reaction was allowed to proceed during one hour and the optical density was measured at 414 nm using an automatic reader. Using the same antiserum (kind gift of Dr. Rokach, Merck Frosst, Canada) at appropriate dilutions (1/30,000 for LTC4 AChE versus 1/6,000 for 3HLTC4) the sensitivities were compared. LTC4 was detectable in the range of 3.3 to 84 femtomoles/well corresponding to a 12–75% displacement of initial binding (i.e. approximately 2–50 pg/well) with LTC4-AChE as compared with 80–1000 pg/tube for 3H. The 50% inhibition was approximately obtained at 15 pg/tube, respectively. The determination of LTC4 on human neutrophils stimulated by various stimuli was performed without any extraction. The results obtained by this technique have been validated by comparing them to those obtained using a quantitative HPLC method. It was also possible to use the same labeling technique for prostaglandin D2-methoxamine, 6-keto PGFlα and TXB2. For all these EIA, the 50% diplacement of initial binding was 2–3 pg/well.     

Effects of thymosin alpha l on some time-dependent functions in thymectomized mice

The effects of a synthetic thymosin alpha1 on the azathioprine-sensitivity of spleen cells and on the thymic-like activity of serum from adult thymectomized mice, were observed. Thymosin alpha1 is able to restore the lowered levels of these T-dependent functions after in vivo administration.     

Circannual rhythm of insulin release and hypoglycemic effect of tolbutamide stimulus in healthy man

Four healthy non obese young volunteers were observed for a 24-hr period, every other month, over the course of one year. Tolbutamide was injected i.v. each day of the experiment every four hours. Tolbutamide-induced insulin secretion (T.I.I.S.) was evaluated by planimetrically measuring insulin areas above basal levels. Tolbutamide-induced hypoglycemic effect was evaluated by measuring the blood glucose difference between the 5th and 25th minute after the drug injection (delta G5'-25'). The macroscopic evaluation of T.I.I.S. and delta G5'-25' (mean chronograms) permitted the detection of the existence of a circannual variation of both variables. In particular the maximum level of the blood glucose drop (delta G5'-25') was registered in February. Subsequently the quantification of the rhythm of T.I.I.S. was obtained by fitting a sine curve, according to the Cosinor method. The highest insulin release was confirmed in winter. As previously documented, the existence of a statistically significant circadian rhythm of T.I.I.S. was confirmed in the morning, i.e. the same period of the day in which insulin-induced hypoglycemia occurs.     

Structural requirements in the reaction of morphine uridine diphosphate glucuronyltransferase with opioid substances.

The influence of the 3-hydroxyl and N-alkyl groups in the reactivity of narcotic compounds with morphine UDP-glucuronyltransferase was studied. Opioids possessing both, one or none of these groups were tested for inhibition of morphine glucuronidation in rabbit liver microsomal preparations. Compounds with only a 3-hydroxyl group (normorphine) or an N-methyl group (codeine, ethylmorphine) were less potent competitive inhibitors than those containing both groups (dextrorphan). Norcodeine, with neither of these groups, had no inhibitory effect. The synthetic narcotics (+)- and (-)-methadone, (-)-alpha-acetylmethadol and meperidine, with only an N-alkyl group, were effective competitive inhibitors. No stereoselectivity of the morphine glucuronyltransferase for opioid isomers was observed, and [methionine]enkephalin does not react with morphine glucuronyltransferase. Differences of pKa values and water/lipid solubility of narcotics could not explain the effects. Results indicate that the N-alkyl group plays a critical role in the interaction of narcotics with the morphine UDP-glucuronyltransferase.     

Capillaries of the adrenal cortex possess aminopeptidase A and angiotensin-converting-enzyme activities.

The enzymes required to convert the prohormone angiotensin I into angiotensins II and III, secretagogues of aldosterone, are enriched in association with capillary endothelium isolated from rat adrenal cortex. Thus the secretion of aldosterone may be controlled, in part, by processing of peptides occurring within the adrenal gland itself.     

Enhancement of α-methylglucoside efflux by respiration in respiratory mutants of Escherichia coli K-12

The rate of α-methylglucoside efflux from wild-type cells of Escherichia coli K-12 is enhanced by different substrates, as long as they are readily respired. A similar enhancement takes place in strains with impaired oxidative phosphorylation (unc mutants), regardless of their being able (strains AN120, N₁₄₄, and AN382) or unable (strain NR70) to energize the membrane through respiratory electron flow. The uncouplers carbonylcyanide-m-chlorophenylhydrazone and tetrachlorosalicylanilide do not diminish the efflux acceleration in wild-type strains or unc mutants. However, the stimulation of α-methylglucoside efflux does not occur in the mutant AN59 which cannot perform a normal respiratory electron transport due to a defective synthesis of ubiquinone. The failure to stimulate the efflux is observed with succinate, which is a typical substrate of respiration, as well as with substrates which can yield ATP both at respiratory and substrate levels such as gluconate or glycerol. Moreover, potassium cyanide nullifies the acceleration of α-methylglucoside efflux caused in any type of strain and by any substrate. These results show that neither ATP nor an energized state of the membrane appears to be needed for respiration to accelerate α-methylglucoside release from E. coli cells, and question the existence of any energy-requiring reaction for αMG exit, previously proposed by other authors.     

The effect of S-adenosyl-L-methionine (SAM) on membrane permeability in the perfused rat liver

S-adenosilmethionine is present in most human tissues and is an important factor for transmethylation, transulphuration and aminopropylation reactions. The compound improves the biological, morphological and histochemical aspects of rat liver following CCl4 intossication. At the same time has been successfully used during chronic liver disease in man. With the aim to better clarify the action mechanism of SAMe some aspects concerning its effects on cell permeability in rat liver, by using the perfusion technique, have been investigated. In particular the capacity of this compound to prevent the enzymatic loss (GPT and GOT) during liver perfusion has been studied. 30 perfusions without SAMe, as control, and 6 by infusing 2 mg of compound during the perfusion time have been accomplished. Varing the perfusion time from 0 to 120 min it has been observed that at any time the presence of the SAMe reduced by about 50% the loss of GOT. Similarly the activity of GPT ranging from 2 to 6 mU/ml indicate that no appreciable enzyme output occurs in presence of SAMe.     

Lectin-mediated sperm agglutination of bufo arenarum and leptodactylus chaquensis spermatozoa

Various plant lecins were employed in cell agglutination experiments to ascertain the presence of specific saccharides in the surface of B arenarum and L chaquensis spermatozoa. B arenarum spermatozoa were specifically agglutinated with Concanavalin A (Con A), phytohemagglutinin P (PHA-P), and wheat germ agglutinin (WGA), but not with soybean agglutinin (SBA). In contrast, L chaquensis spermatozoa were strongly agglutinated by SBA, WGA, and PHA-P. L chaquensis spermatozoa did not agglutinate with Con A even at high concentrations. Lectinmediated sperm agglutination was inhibited in the presence of specific lectinbinding sugars. Spermatozoa from both species were agglutinated randomly with all lectins suggesting a uniform distribution in the sperm surface of the lectinbinding saccharide ligands. B arenarum sperm agglutination induced by Con A is sensitive to temperature. B arenarum spermatozoa are more agglutinable at 24°C than at 4°C. These results suggest that lectin-binding site mobility is necessary for sperm agglutination.     

Superficial mycoses in Italy

A total of 1244 cases suspected of superficial mycoses were analysed in a period of 12 months in Italy. Dermatophytoses were found in 62.4%; the other common disease was Pityriasis versicolor (44.1%). The predominant speciesEpidermophyton floccosum (34,2%) was strickingly high in relation to other statistics; this species was commonest in tinea cruris. Among the other dermatophytesM. canis was predominant (31.3%) commonest in tinea corporis, followed byT. mentagrophytes (17,8%) andT. rubrum (10,3%). A high rate of yeasts was found in tinea manum (81.6%) and tinea pedis (47.7%).