Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450

Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide).     

Techniques for encapsulating bioactive agents into liposomes

As a prerequisite for the use of liposomes for delivery of biologically active agents, techniques are required for the efficient and rapid entrapment of such agents in liposomes. Here we review the variety of procedures available for trapping hydrophilic and hydrophobic compounds. Considerations which are addressed include factors influencing the choice of a particular liposomal system and techniques for the passive entrapment of drugs in multilamellar vesicles and unilamellar vesicles. Attention is also paid to active trapping procedures relying on the presence of (negatively) charged lipid or transmembrane ion gradients. Such gradients are particularly useful for concentrating lipophilic cationic drugs inside liposomes, allowing trapping efficiencies approaching 100%.     

In vivo formation of sigma-methyl- and sigma-phenyl-ferric complexes of hemoglobin and liver-cytochrome P-450 upon treatment of rats with methyl- and phenylhydrazine

Ferric sigma-phenyl complexes of hemoglobin and liver cytochrome P-450 are formed in vivo upon administration of C6H5NHNH2 to rats. Small amounts of the sigma-methyl complex of hemoglobin were also detected in vivo upon treatment of rats with CH3NHNH2. At the doses used for CH3NHNH2 (25 and 50 mg/kg) the states and levels of hemoglobin in the blood and spleen, and of cytochrome P-450 in the liver were almost unchanged. On the contrary, C6H5NHNH2 (25-100 mg/kg) led to a decrease of the HbO2 blood level (10-50%), together with an increase in the HbFe(III) level and the appearance of the HbFe(III)-C6H5 complex. The concentration of this complex reaches its maximum value (2 mM) 1 h after C6H5NHNH2 administration (20% of total hemoglobin). At the same time large amounts of HbO2, HbFe(III) and HbFe(III)-C6H5 appeared in the spleen, and remained high up to 24 h after treatment. Treatment of rats with C6H5NHNH2 (25-100 mg/kg) led to a significant decrease in the level of liver cytochrome P-450 (a 70% decrease 2 h after treatment with 100 mg/kg C6H5NHNH2). About 15% of the remaining cytochrome P-450 existed as a cyt.-P-450-Fe(III)-C6H5 complex, a new example of cytochrome P-450-Fe-metabolite complex which is stable in vivo.     

Platelet serotonin content and uptake in spontaneously hypertensive rats

Platelet serotonin (5-HT) content and uptake were studied in male SHR and WKY at various ages. Blood was withdrawn from the carotid artery under anesthesia and 5-HT levels determined from platelet rich plasma (PRP) using a HPLC technique coupled with an electrochemical detection method. Platelet 5-HT uptake was studied by incubating PRP at 37 degrees C for 10 sec with increasing concentrations of 3H-5HT. Lineweaver- Burk plots of 3H-5HT uptake were linear suggesting simple Michaelis- Menten uptake kinetics. The SHR had more platelets than age-matched controls and consequently a higher blood circulating pool of 5-HT. Nevertheless, the 5-HT platelet levels were similar to those of their age-matched rats. The 5 week-old SHR and WKY had greater numbers of platelets and higher 5-HT platelet levels than the older rats of both strains. The affinity constants (Km) and the maximal velocities (Vmax) of platelet 5-HT uptake did not differ significantly between the 12 week- and the 6 month-old SHR and WKY. These data suggest that the SHR do not show the same impairment in platelet 5-HT metabolism as observed in essential hypertension in man.