[Familial Alzheimer's disease mutations in the presenilin 1 gene reduce cell-cell adhesion in transfected fibroblasts].

Experimental evidence has been obtained that mutations in the presenilin 1 (PS1) gene in familial Alzheimer's disease can lead to the disturbance of cell adhesion in model cell cultures. It was shown that, in L fibroblasts of mice with stable expression of GFP-PS1 cDNA containing G209V or E319G mutations, cell-cell interactions and the accumulation of GFP-PS1 cDNA in intercellular contacts are disturbed. Similar results were obtained in transfected human epithelial Hep2 cells. It is assumed that mutations in familial Alzheimer's disease lead to the disturbance of the functions of presenelin 1 in cell adhesion.

Study of interaction of ceruloplasmin with serprocidins.

This paper describes formation of complexes of ceruloplasmin (CP) with such proteins of the serprocidin family as azurocidin (CAP37), neutrophilic elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). We present evidence that serprocidins form complexes with CP at a molar ratio 1 : 1. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, did not prevent the interaction of serprocidins with CP in the course of SDS-free disc electrophoresis. CP affected the activities of NE, CG, and PR3 as a competitive inhibitor with K(i) ~ 1 µM. Inhibitory effect of CP depended on ionic strength of the solution and was negligible at NaCl concentrations above 300 mM. In the mode of competitive inhibitors serprocidins suppressed oxidase activity of CP towards p-phenylenediamine. CAP37 displayed the strongest inhibitory effect (K(i) ~ 20 nM). Upon adding various serprocidins to human, rat, rabbit, dolphin, dog, horse, and mouse plasma only CAP37 would form a complex with CP. Synthetic peptide RKARPRQFPRRR (5-13, 61-63 CAP37) displaced CAP37 from its complex with CP. Adding CAP37 to the triple complex formed by CP, lactoferrin, and myeloperoxidase resulted in displacement of the latter from the complex. The dissociation constant of CAP37 with immobilized CP was 13 nM. Therefore, among serprocidins CAP37 can be regarded as the specific partner of CP.

[Degeneration of growth cones in a culture of embryonic neurons of mouse with presenilin 1 gene knockout].

A comparative study of growth cone morphology in cultured embryonic neurons derived from wild type PS 1(+/+) and knockout PS 1(-/-) mice has been performed. Growth cones from wild type PS 1(+/+) mice were well spread and usually formed radially continuous and regular lamellar extensions with numerous filopodia. In contrast, most growth cones from knockout PS 1(-/-) mice exhibited small lamellar extensions, short filopodia, and pure adhesion. A significant amount of growth cones from knockout PS 1(-/-) mice collapsed after 3-4 days in culture. It was suggested that PS 1 plays an important role in growth cone structure by stabilizing the integrity of the cytoskeleton. The growth cone collapse may be the main reason for abnormal neuronal migration and impaired synaptic function in PS 1(-/-) mice.

Identification of leukocyte cationic proteins that interact with ceruloplasmin.

Proteins from leukocytes were investigated for their ability to interact with ceruloplasmin (Cp), a copper-containing glycoprotein of human plasma. Extract from leukocytes was subjected to affinity chromatography on Cp-Sepharose, after which proteins were eluted from the resin with 0.5 M NaCl in Tris-HCl, pH 7.4. SDS-PAGE of the eluate revealed protein bands with molecular weights 78, 57, 40, 30, 16, and 12 kD. Among these, Western blotting detected myeloperoxidase (57, 40, and 12 kD) and lactoferrin (78 kD). Also, the 30-kD component had a sequence (1)I-(2)I/V-(3)G-(4)G-(5)R/H at the N-terminus that is likely to indicate the presence of neutrophilic elastase, cathepsin G, proteinase 3, and azurocidin (CAP 37) - all from the family of serprocidins. Mass spectrometry of tryptic fragments indicated the presence of the 16-kD eosinophilic cationic protein (seven peptides), 27-kD cathepsin G (eleven peptides), 27-kD azurocidin (eight peptides), 29-kD neutrophilic elastase (seven peptides), and 27-kD proteinase 3 (six peptides). Myeloperoxidase was represented by 57-, 40-, and 12-kD fragments (thirteen, ten, and four peptides, respectively). Thus, interaction with Cp of five cationic proteins, i.e. of eosinophilic cationic protein, cathepsin G, neutrophilic elastase, proteinase 3, and azurocidin is reported for the first time.

[Expression of presenilin 1 on the cell surface in motile polarized cells].

Most cases of familial early-onset Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene. However, the cellular functions of PS1 are not yet completely understood. We showed that endogenous PS1 and the adhesion protein CD44 are redistributed on the surface of cell projections (lamellipodia) in polarized T- lymphocytes (Jurkat cells) after the adhesion to a collagen matrix. This effect was not observed for another surface protein of T lymphocytes, which is not involved in cell adhesion processes, the T cell receptor. In primary cultures of mouse cortical neurons, PS1 was concentrated at the surface of extended growth cones and at the sites of neurite contacts. The concentration of PS1 at the surface of cellular structures that promote cell motility and cell contacts suggests an important role of PSI in cell adhesion in motile polarized cells.

Identification and isolation from breast milk of ceruloplasmin-lactoferrin complex.

The presence of a complex of the copper-containing protein ceruloplasmin (Cp) with lactoferrin (Lf) in breast milk (BM) is shown for the first time. In SDS-free polyacrylamide gel electrophoresis (PAGE), electrophoretic mobility of Cp in BM is lower than that of plasma Cp, coinciding with the mobility of the complex obtained upon mixing purified Cp and Lf. Affinity chromatography of delipidated BM on Cp-Sepharose resulted in retention of Lf. SDS-PAGE of the 0.3 M NaCl eluate revealed a single band with Mr approximately 78,000 that has the N-terminal amino acid sequence of Lf and reacts with antibodies to that protein. Synthetic peptides R-R-R-R (the N-terminal amino acid stretch 2-5 in Lf) and K-R-Y-K-Q-R-V-K-N-K (the C-terminal stretch 29-38 in PACAP 38) caused efficient elution of Lf from Cp-Sepharose. Cp-Lf complex from delipidated BM is not retained on the resins used for isolation of Cp (AE-agarose) and of Lf (CM-Sephadex). Anionic peptides from Cp--(586-597), (721-734), and (905-914)--provide an efficient elution of Cp from AE-agarose, but do not cause dissociation of Cp-Lf complex. When anti-Lf is added to BM flowed through CM-Sephadex, Cp co-precipitates with Lf. Cp-Lf complex can be isolated from BM by chromatography on CM-Sephadex, ethanol precipitation, and affinity chromatography on AE-agarose, yielding 98% pure complex. The resulting complex Cp-Lf (1 : 1) was separated into components by chromatography on heparin-Sepharose. Limited tryptic hydrolysis of Cp obtained from BM and from blood plasma revealed identical proteolytic fragments.

[Protein of Escherichia coli interacting specifically with human low density lipoproteins]. / Belok kishechnoi palochki, spetsificheski vzaimodeistvuiushchii s lipoproteinami nizkoi plotnosti cheloveka.

Escherichia coli 48 kDa protein interacting specifically with human low-density lipoproteins is described. The dissociation constant of this highly specific interaction was found to be equal to 4 mkg LDL per 1 ml or 7.3 x 10 M, which is comparable with the dissociation constant of the complex formed by LDL and human LDL receptor. A protocol for purifying the E. Coli binding protein was developed and antibodies against this purified protein were raised. The absence of sequences with homology to the ligand-binding repeats of the human LDL receptor in E. Coli proteome was shown by computer analysis of E. Coli genome. A conclusion was made that binding of the human LDL with specific E. Coli protein is thus mediated by other sequences and by another mechanism different from that, which occurs in human cells during the interaction of lipoproteins with their specific receptor. The establishment of specific interaction between E. Coli protein and human LDL can turn out to be useful in the future for purifying lipoproteins of a specific class and for administering plasmapheresis in patients with severe hyperlipoproteinemia.

Expression of cDNA fragment encoding ligand-binding domain of human low density lipoprotein receptor in Escherichia coli cells.

To study structure-function relationships in low density lipoprotein receptor (LDLR), a key protein in human cholesterol metabolism, it is reasonable to operate with separate protein domains. To obtain highly purified functionally active LDLR ligand-binding domain, we have cloned the corresponding LDLR cDNA fragment in two expression plasmid vectors of Escherichia coli. We have developed methods to purify fusion and practically individual recombinant proteins and characterized the obtained products biochemically. Antibodies raised against fused with beta-galactosidase and individual recombinant protein have been shown to be efficient in identification of LDLR protein in crude lysates of human fibroblasts (cell line HT-1080).

[The modelling of the genetic correction of familial hypercholesterolemia by creating cellular clones stably expressing the human low-density lipoprotein receptor]. / Modelirovanie geneticheskoi korrektsii semeinoi giperkholesterinemii putem sozdaniia kletochnykh klonov, stabil'no ékspressiruiushchikh retseptor lipoproteinov nizkoi plotnosti cheloveka.

The stable expression of human low-density lipoprotein receptor (rLDL) was studied in Chinese hamster ovary (CHO) cells transformed by the pMSVL recombinant plasmid. This plasmid contains full-length rLDL cDNA and hybrid promoter consisting of regulatory elements from early SV-40 promoter and glucocorticoid responsive enhancer of Moloney sarcoma virus. The expression of rLDL cDNA in transformed cells was demonstrated using RNA-cDNA blot hybridization, PCR analysis, and immunofluorescent analysis.