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.     

Structure-function analysis of plasma cholesteryl ester transfer protein by protease digestion and expression of cDNA fragments in Escherichia coli

In an attempt to define an active domain of the protein, fragments of cholesteryl ester transfer protein (CETP) were obtained by limited digestion of the native, plasma-derived protein with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease or by expression of CETP cDNA restriction fragments in Escherichia coli. Although digestion of native CETP with these proteases resulted in extensive fragmentation of the protein and loss of the intact 74-kDa molecule as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CE transfer activity was unaffected (trypsin or chymotrypsin treatment) or only partially lost (V8 protease treatment). Analysis by molecular sieve chromatography showed that the CE transfer-active product of this proteolysis consisted of polypeptide fragments which remained associated, retaining the native molecular weight of CETP. These proteolyzed complexes were resistant to dissociation by dithiothreitol, 8 M urea, or delipidating agents. As shown by CE transfer activity, native CETP was found to possess a stable conformation which remained unchanged in buffers containing up to 4.5 M urea, or following exposure to even higher (8 M) urea concentrations. CETP polypeptides from bacterially expressed cDNA fragments were found to be catalytically inactive although they contained the epitope for an inhibitory anti-CETP monoclonal antibody and had emulsion binding properties similar to native CETP. Selected synthetic CETP peptides (including the peptide containing the inhibitory monoclonal antibody epitope) were also devoid of CE transfer activity. Thus, no evidence was found for an independently active subunit of the CETP. Together, the results indicate that the CETP possesses a distinct and highly stable tertiary structure which is required for CE transfer catalytic activity.     

Bioguided Fractionation and Isolation of an Antiplasmodial Saponin from the Roots of Nauclea xanthoxylon (A.Chev.) Aubrév. (Rubiaceae)

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC₅₀s) of 0.57 and 1.26 μg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC₅₀s of 2.68 and 1.85 μg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside ( 1 ) with IC₅₀s of 0.33 and 1.30 μM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid ( 2 ), ursolic acid ( 3 ), quafrinoic acid ( 4 ), quinovic acid ( 5 ), quinovic acid 3-O-β-D-fucopyranoside ( 6 ), oleanolic acid ( 7 ), oleanolic acid 3-acetate ( 8 ), friedelin ( 9 ), β-sitosterol ( 10a ), stigmasterol ( 10b ) and stigmasterol 3-O-β-D-glucopyranoside ( 11 ). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside ( 1 ) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria.     

Kinetic parameters of lactate dehydrogenases of some rumen bacterial species,the anaerobic ciliate Isotricha prostoma and mixed rumen microorganisms

A number of kinetic parameters of the lactate dehydrogenases of three rumen bacterial species (Peptostreptococcus productus, Propionibacterium acnes and Actinomyces viscosus), the rumen ciliate Isotricha prostoma and mixed rumen microorganisms (MRM) with respect to NADH, pyruvate, fructose-1,6-diphosphate (FDP) as well as the effects of several nucleotide phosphates were studied.Partially purified LDH of Peptostr. productus had the same kinetic parameters as in crude cell free extracts. Values for K_m, determined by Michaelis-Menten kinetics with pyruvate as the substrate, were in the same range for all lactate dehydrogenases. After feeding a cow, changes in the apparent K_m and V_max values for NADH of the total LDH activity in MRM were followed.It is suggested that of the factors studied the ratio NADH/NAD(H) and ATP are the most important regulatory factors for the lactate dehydrogenases of mixed rumen microorganisms.The investigation were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO)