Isolation and characterization of sheep lactoferrin, an inhibitor of platelet aggregation and comparison with human lactoferrin.

Highly purified sheep lactoferrin was isolated from ovine whey in a single chromatographic step (FPLC): it was characterized by electrophoresis, N-terminal sequence determination and compared with lactoferrins from other species. Sheep and human lactoferrins inhibited thrombin-induced platelet aggregation (median inhibitory concentration: IC50 5 and 4 microM, respectively). Pepsin hydrolysates of human and sheep lactoferrins were fractionated by reverse-phase high-performance liquid chromatography and only one peak was an inhibitor of platelet aggregation. The sheep or human lactoferrin binding to platelets was studied.

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.

Sheep kappa-casein peptides inhibit platelet aggregation.

The C-terminal part (residues 106-171) of sheep kappa-casein, called caseinoglycopeptide (CGP), inhibits thrombin- and collagen-induced platelet aggregation in a dose-dependent manner (mean inhibitory concentration (IC50) 215 microM and 100 microM, respectively). An enzymatic hydrolysate of CGP was fractionated by reverse phase high performance liquid chromatography: three peptides KDQDK (residues 112-116), TAQVTSTEV (residues 163-171) and QVTSTEV (residues 165-171) completely inhibited thrombin-induced platelet aggregation. CGP at a concentration near its IC50 had a very long life when incubated in human or guinea-pig plasma. An ex vivo experiment showed that 17% of CGP was found 60 min after its i.v. bolus injection in guinea-pig. By hydrophobic cluster analysis, human fibrinogen and sheep kappa-casein peptides, inhibitors of platelet aggregation, were compared and we observed similarities for their C-terminal parts and for their short peptides (RGDF and KDQDK).

Genetic architecture of testis and seminal vesicle weights in mice.

Comparisons across 13 inbred strains of laboratory mice for reproductive organ (paired seminal vesicles and paired testes) weights indicated a very marked contrast between the C57BL/6By and NZB/BINJ mice. Subsequently these strains were selected to perform a quantitative genetic analysis and full genome scan for seminal vesicle and testis weights. An F(2) population was generated. The quantitative genetic analyses indicated that each was linked to several genes. Sixty-six short sequences for length polymorphism were used as markers in the wide genome scan strategy. For weight of paired testes, heritability was 82.3% of the total variance and five QTL contributed to 72.8% of the total variance. Three reached a highly significant threshold (>4.5) and were mapped on chromosome X (LOD score 9.11), chromosome 4 (LOD score 5.96), chromosome 10 (LOD score 5.81); two QTL were suggested: chromosome 13 (LOD score 3.10) and chromosome 18 (LOD score 2.80). Heritability for weight of seminal vesicles was 50.7%. One QTL was mapped on chromosome 4 (LOD score 9.21) and contributed to 24.2% of the total variance. The distance of this QTL to the centromere encompassed the distance of the QTL linked with testicular weight on chromosome 4, suggesting common genetic mechanisms as expected from correlations in the F(2). Both testis and seminal vesicle weights were associated with a reduction in the NZB/BINJ when this strain carried the Y(NPAR) from CBA/H whereas the Y(NPAR) from NZB/BINJ in the CBA/H strain did not modify reproductive organ weights, indicating that the Y(NPAR) interacts with the non-Y(NPAR) genes. The effects generated by this chromosomal region were significant but small in size.

Immunostimulating properties and three-dimensional structure of two tripeptides from human and cow caseins.

Some tripeptides obtained by enzymic digestion of caseins possess immunomodulating properties. In order to correlate activity and structure, X-ray analysis has been applied to two of them Leu-Leu-Tyr and Gly-Leu-Phe.

Casein peptide release and passage to the blood in humans during digestion of milk or yogurt.

In adult humans, after milk or yogurt ingestion, many peptides derived from alpha s1-, beta- or kappa-caseins were detected in stomach, including the kappa-caseinoglycopeptide, an inhibitor of platelet aggregation. Smaller peptides derived from casein and lactoferrin were recovered from duodenum. Two long peptides, the kappa-caseinoglycopeptide and the N-terminal peptide of alpha s1-casein, were absorbed and detected in plasma. These results support the concept that food-born peptides could have physiological activities in man.

Enhancement of phagocytic activity of human monocytic-macrophagic cells by immunostimulating peptides from human casein.

Two immunostimulating peptides, Val-Glu-Pro-Ile-Pro-Tyr and Gly-Leu-Phe, obtained from human caseins, were demonstrated to significantly stimulate binding of human senescent red blood cells to human monocytic-macrophagic cells and their phagocytosis by these cells.

Effects of tripeptides derived from milk proteins on polymorphonuclear oxidative and phosphoinositide metabolisms.

The tripeptide GLF (glycyl-leucyl-phenylalanine) was isolated from human milk proteins. This peptide increased phagocytosis by human and murine macrophages and protected mice against Klebsiella pneumoniae infection. Specific binding sites on human polymorphonuclear leukocytes (PMNs) have been demonstrated recently. The aim of the present research was to study the action of this peptide on rat and human PMN oxidative burst and to investigate the consequences of cell stimulation on polyphosphoinositide hydrolysis. A biphasic stimulating concentration-dependent effect of GLF on PMN chemiluminescence and superoxide anion generation was demonstrated. One of the peaks of the oxidative response occurred around 10(-9) M, which correlates with the Kd of high affinity receptors of GLF. The other maximum, around 10(-4) M, might be due to the hydrophobic nature of the tripeptide. O2- generation mimicked the phorbol myristate acetate response: after a lag period of 2-5 min, O2- release gradually increased for 10-15 min until a plateau was reached. Furthermore, GLF enhanced phosphoinositide breakdown with maximal IP3 production at 10(-7) M. Various analogs of GLF were synthesized in order to define the relative importance of the different amino acids and their position in the tripeptide molecule: glycyl-phenylalanine-leucine was devoid of biological properties but enhanced the activity of GLF on the metabolic burst at high concentrations; peptides leucyl-leucyl-phenylalanine and leucyl-leucyl-tyrosine, which displaced GLF from its specific membrane receptors, exerted stimulating effects on PMN oxidative and phosphoinositide metabolisms. It is quite conceivable that these short peptides, which may be generated in the newborn during digestion and which are able to stimulate phagocytic cells, are implicated in the defense of the neonate immature organism against infection.

Casein, a prohormone with an immunomodulating role for the newborn?

Maternal colostrum and milk, the earliest food of the newborn, should not only be considered as supplying nutrients, but also as agents providing protection against aggressions from the new environment. Indeed by enzymatic digestion of the main milk proteins, the caseins, biologically active peptides are released; they may be implicated in the stimulation of the newborn's immune system. From this point of view a 'strategic active zone' has been characterized in beta-casein. A possible role of casein as a 'prohormone' for the newborn is suggested.

Biologically active casein peptides implicated in immunomodulation.

Maternal milk should not only be considered as a nutrient, but also as a protecting agent against aggressions from the neonate's new environment. Breast-feeding facilitates transmission of a passive immunity by multifunctional factors which have a direct effect on the neonate's resistance to bacterial and viral infections. Among these factors are the main milk proteins, the caseins: during enzymic digestion of human and bovine caseins, immunomodulating peptides are released. Corresponding synthetic peptides stimulated in vitro phagocytic activity of murine and of human macrophages and exerted in vivo a protective effect against Klebsiella pneumoniae infection of mice. These data suggest that casein peptides may exert a stimulating function on the immune system of the newborn.

Induction of allergic encephalomyelitis using hydrosoluble mycobacterial fractions.

Experimental allergic encephalomyelitis has been induced in guinea pigs employing bovine myelin basic protein as the antigen and a hydrosoluble tetrasaccharide-heptapeptide from delipidated cells of the human mycobacterial strain H37Ra as adjuvant. The maximum response was observed using 33 mug of antigen and 12.5 mug of adjuvant per animal.

Induction of allergic encephalomyelitis using hydrosoluble adjuvant and the tryptophan region of myelin basic protein.

Experimental allergic encephalomyelitis has been induced in guinea pigs using the encephalitogenic tryptophan peptide as antigen and a hydrosoluble adjuvant extracted from Mycobacterium tuberculosis, var. hominis, strain H37Ra. The maximum response was observed using 100mug of adjuvant per animal. This is a quantity of adjuvant substantially higher than was necessary to induce disease utilizing the whole myelin basic protein as antigen.

A new enzymatic pathway of citrullinogenesis in murine hemopoietic cells.

Citrullinogenesis is demonstrated when murine bone marrow cells are incubated with dialyzed secondary mixed leukocyte culture supernatant. The identity of citrulline in bone marrow cell supernatants has been established by gas chromatographic mass spectrometric analysis. It is shown that, in our model, citrulline synthesis proceeds directly from arginine without intermediate ornithine production, ruling out the involvement of ornithine transcarbamylase (EC 2.1.3.3.). Moreover, none of the other enzymatic activities described for catalyzing citrullinogenesis, i.e. arginine deiminase or peptidyl arginine deiminase can be demonstrated. The generation of oxygen radicals is necessary for this enzymatic reaction. It is induced by a thermolabile protein produced during the antiallograft immune response with a molecular weight of about 150,000.

Cytochrome P450 4A11 expression in human keratinocytes: effects of ultraviolet irradiation.

BACKGROUND: The skin is the major interface between the body and its environment. Directly and continuously exposed to a large variety of foreign agents and stimuli such as ultraviolet radiation (UVR), cutaneous cells are active sites of intense metabolism. The cytochromes P450 (P450) are a group of enzymes that play an important part in the protective role of the skin; they are a family of microsomal membrane-bound mono-oxygenases. These haem-containing proteins catalyse the insertion of an atom of molecular oxygen into the substrate. Although generally present at low levels, a certain number of these enzymes have now been characterized in mammalian skin as constitutive or inducible isoforms. OBJECTIVES: To test the effects of UVR, a source of oxidative stress, on the expression of mRNA coding for several P450 isoforms (CYP), with particular reference to the CYP2E1 and CYP4A11 isoforms, which might play a role in lipid metabolism in human keratinocytes. METHODS: Human keratinocytes were cultured, irradiated and mRNA expression was analysed by gel electrophoresis after reverse transcriptase polymerase chain reactions. CYP proteins were determined from keratinocyte microsomal fractions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoperoxidase staining. Thin layer chromatography was used to detect (omega-1)- and (omega)-hydroxylation of lauric acid in the microsomal fractions. RESULTS: mRNAs for CYP2E1, CYP1A1 and CYP3A5 were expressed in all the keratinocyte preparations tested; however, neither CYP3A4 nor CYP3A7 were detected, either in the presence or absence of UVR treatment. CYP19Aro, CYP2C19 and CYP26 were not expressed constitutively, although some induction of CYP19Aro was seen after combined UVB and UVA irradiation. CYP4A11 mRNA was not detected in any keratinocyte preparations either under control conditions or after UVB treatment. Nevertheless, in non-irradiated keratinocyte microsomes, two protein bands were immunoreactive with anti-CYP4A11 enzyme antibodies, one of which corresponds to CYP4A11 protein. UVA treatment of cultured keratinocytes induced CYP4A11 mRNA expression after 24 h, as well as an increase in immunoreactivity of the two protein bands. Although (omega-1)- and (omega)-hydroxylation of fatty acids is attributed to CYP2E1 and CYP4A11, respectively, in the liver or kidney, no omega-hydroxylation of lauric acid was observed in microsomal preparations from cultured keratinocytes. CONCLUSIONS: However, CYP4A11 may participate in the defence mechanism against UVA-induced oxidative damage.

In vivo effects of immunostimulating lipopeptides on mouse liver microsomal cytochromes P-450 and on paracetamol-induced toxicity.

Immunomodulating lipopeptides lauroyl-L-Ala-gamma-D-Glu-LL-A2pmNH2-Gly (RP 44.102) and lauroyl-L-Ala-gamma-D-Glu-LL-A2pmNH2 (RP 56.142) were found to protect mice against the hepatotoxicity of paracetamol, which is due to cytochrome P-450 dependent formation of toxic metabolites and radicals. In fact they decreased the amount of hepatic microsomal cytochrome P-450, and the level of CCl4-induced lipid peroxidation. In contrast lauroyl-L-Ala-gamma-D-Glu-DD-A2pmNH2 (RP 53.204), which only differs by the configuration of the two chiral carbons of A2pm (diaminopimelic acid) and is not an immunomodulating agent, failed to protect against poisoning by paracetamol and had no effect on the level of hepatic cytochrome P-450 or the microsomal CCl4-induced lipid peroxidation. This provides a clear connection between the immunostimulating properties of a compound and its effects on xenobiotic biotransformations.

Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin.

A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.

Binding of the bovine caseinoglycopeptide to the platelet membrane glycoprotein GPIb alpha.

The bovine caseinoglycopeptide (residues 106-169), the C-terminal part of kappa-casein, inhibited the von Willebrand factor-dependent platelet aggregation in a dose-dependent manner. An affinity matrix made of the caseinoglycopeptide selectively bound the platelet membrane glycoprotein GPIb alpha which contains the von Willebrand factor binding site. The amino acid residues of GPIb alpha participating in the caseinoglycopeptide binding were located after residue Glu 90.