Identification of the major endogenous leukotriene metabolite in the bile of rats as N-acetyl leukotrience E4

Mercapturic acid formation, an established pathway in the detoxication of xenobiotics, is demonstrated for cysteinyl leukotrienes generated in rats after endotoxin treatment. The mercapturate N-acetyl-leukotriene E₄ (N-acetyl-LTE₄) represented a major metabolite eliminated into bile after injection of [³H]LTC₄ as shown by cochromatography with synthetic N-acetyl-LTE₄ in four different HPLC solvent systems. The identity of endogenoud N-acetyl-LTE₄ elicited by endotoxin was additionally verified by enzymatic deacetylation followed by chemical N-acetylation. The deacetylation was catalyzed by penicillin amidase. Endogenous cysteinyl leukotrienes were quantified by radioimmunoassay after HPLC separation. A N-acetyl-LTE₄ concentration of 80 nmol/l was determined in bile collected between 30 and 60 min after endotoxin injection. Under this condition, other cysteinyl leukotrienes detected in bile by radioimmunoassay amounted to less than 5% of N-acetyl-LTE₄. The mercapturic acid pathway, leading from the glutathione conjugate LTC₄ to N-acetyl-LTE₄, thus plays an important role in the deactivation and elimination of these potent endogenous mediators.     

Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

Reduced mechanical activity of perfused rat heart following morphine or enkephalin peptides administration

In the isolated and perfused rat heart, the addition of morphine, methionine-enkephalin or leucine-enkephalin to the coronary perfusate, significantly reduces the mechanical activity by negatively affecting both the heart rate and the developed tension. These effects are dose dependent and maximally evident with leucine-enkephalin. Furthermore all the opioids strongly reduce the activity of isoproterenol-stimulated hearts. The suggestion is made that opioid peptides directly influence the cardiac mechanical activity possibly by interacting with membrane-receptor systems.     

Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.     

Identifiability from parameter bounds. Structural and numerical aspects

Some a priori and a posteriori aspects of the identifiability problem for unidentifiable models are discussed. It is argued that the nation of identifiability from parameter bounds has a minor a priori structural relevance. The parameter bounds rationale may prove a useful a posteriori numerical notion. However, its practical potentiality needs careful evaluation, as the use of point estimates automatically builds into the model some hidden structural constraints. Examples are given.     

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.     

Origin of the Thymidine Kinase Induced by Polyoma Virus in Productively Infected Cells

Cells of the 3T3 mouse line efficiently supported the multiplication of polyoma virus, and the infectious process was accompanied by a marked increase in thymidine kinase (TK) activity. Two lines of 5-bromodeoxyuridine-resistant 3T3 cells have been isolated. As expected, these cells incorporated practically no exogenous thymidine into their deoxyribonucleic acid (DNA) and contained negligible TK activity. Like the parental 3T3 cells, TK(-) lines were susceptible to productive infection by polyoma virus, but infection did not lead to an increase in TK activity. Since kinase activity did appear after infection with another virus (vaccinia) known to contain the gene(s) for that enzyme, it is concluded that TK is not one of the gene products of polyoma virus. As induction of cellular DNA synthesis by polyoma virus occurs normally when the TK(-) cells are infected in the stationary phase, TK cannot play a role in the determination of this phenomenon.     

The ATP-bioluminescence method for a rapid evaluation of the microbial activity in the stone materials of monuments

The examination of the state of conservation of works of art in stone includes the assessment of the presence of microbiological agents on the surface of the decayed monuments. These microorganisms can accelerate, via their metabolic activity, the decay process of the stone surface. At present this assessment is made with the traditional techniques for the microbiological examination of the soil, provides results only after a delay of 30 days. A bioluminescent ATP assay should provide rapid quantitation of actively growing organisms on the surface of a stone monument, and the applicability of this technique was verified on some samples of sandstone (Pietraforte) collected from a historic building (the Strozzi Palace) in Florence. These samples were evaluated for the amount of the ATP and the total number of microorganisms. The results obtained suggest that the bioluminescent assay could be suitable for detecting and quantitating the presence of microorganisms in a sample of stone.     

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 M benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.