Expression and modulation of the Lewis x antigen (CD15) on the T cell line Molt-4.

The T cell lines Molt-4 and H9 exhibited a characteristic distribution of the cell adhesion molecule Lewis x (CD15, lacto-N-fucopentanose III) showing an unusually broad peak by flow cytometry ranging from cells without CD15 to cells with high expression. The cytokines IL-1, IL-2, IFN-beta, IFN-gamma, and TNF-alpha, known to activate T cells, did not affect CD15 expression. However, phorbol myristate acetate and the thymic peptide extract Thymex-L were able to enhance both the number of CD15-positive cells and the median fluorescence. The effects of both inducers were dose- and time-dependent. An additive or synergistic effect was not seen. These data suggest that phorbol esters and distinct thymic peptides can modulate the expression of the cell surface antigen CD15.

Prothymosin alpha 1 effects in vitro on chemotaxis, cytotoxicity and oxidative response of neutrophils from melanoma, colorectal and breast tumor patients.

Immunoregulatory effects of thymic peptides on functions of polymorphonuclear leukocytes (PMNs) are poorly investigated. We studied the effects of prothymosin alpha 1 (Pro alpha 1) on PMNs from patients with colorectal tumors, breast tumors and melanoma (total n = 37) in comparison with healthy donors (n = 18), with respect to chemotaxis, cytotoxicity against HCT-116 colon tumor cells, oxidative response (chemiluminescence reaction) as well as expression of surface marker molecules. We found that Pro alpha 1 was equally effective in stimulating the chemotactic activity of PMNs from tumor patients and healthy donors (43% increase). PMNs from tumor patients, especially with breast tumor, showed a significant enhancement of cytotoxicity against the tumor target cells in comparison with healthy donors. With respect to the PMNs cytotoxicity, only about 50% of the colorectal tumor patients and healthy donors responded to Pro alpha 1 and FMLP. As to the oxidative response of PMNs, elevated levels were found only among colorectal tumor patients. Pro alpha 1 significantly increased the oxidative response in breast and colorectal tumor patients by 55% and 25%, respectively. Pro alpha 1 decreased the expression of CD16 on PMNs of healthy donors, but not that of CD11a, CD11b, CD11c, CD13, CD14, CD15 and CD32. Therefore, we suggest, that Pro alpha 1 may improve some PMN functions of tumor patients, associated with the proposed role in host-tumor interaction.

Cytotoxicity of solid lipid nanoparticles as a function of the lipid matrix and the surfactant.

PURPOSE: Assessment of the in vitro cytotoxicity of solid lipid nanoparticles (SLNs) as a function of lipid matrix (Dynasan 114, Compritol ATO 888), and stabilizing surfactant (poloxamers, Tween 80, soya lecithin, and sodium dodecyl sulphate). Comparison with other colloidal carriers should determine their potential use in the clinic. METHODS: SLNs were produced by high pressure homogenisation. Cytotoxicity was assessed by measuring the viability of HL60 cells and human granulocytes after incubation with SLNs. Particle internalisation was quantified by chemiluminescence measurements. RESULTS: The nature of the lipid had no effect on viability; distinct differences were found for the surfactants. Binding to the SLN surface reduced markedly the cytotoxic effect of the surfactants, e.g., up to a factor of 65 for poloxamer 184. The permanent HL60 cell line-differentiated from cells with granulocyte characteristics by retinoic acid treatment-yielded results identical to freshly isolated human granulocytes. In general, the SLNs showed a lower cytotoxicity compared to polyalkylcyanoacrylate and polylactic/glycolic acid (PLA/ GA) nanoparticles. CONCLUSIONS: Because the results are identical when using human granulocytes, differentiated HL60 cells can be used as an easily accessible in vitro test system for i.v. injectable SLN formulations. The SLNs appear suitable as a drug carrier system for potential intravenous use due to their very low cytotoxicity in vitro.

Prothymosin alpha: a biologically active protein with random coil conformation.

Prothymosin is an acidic protein with an unusual amino acid composition. Though its exact function is not yet known, its high evolutionary conservation and wide tissue distribution suggest an essential biological role. Its physical state, which is controversially discussed in previous publications, was investigated using small-angle X-ray scattering, dynamic light scattering, mass spectrometry, and circular dichroism (CD). Our results unequivocally demonstrate that prothymosin is a monomer under physiological conditions. The protein adopts a random coillike conformation but exhibits persistence of direction and curvature. No regular secondary structure is detectable by CD. The Stokes radius, Rs = 3.07 nm, and the radius of gyration, RG = 4.76 nm, are 1.77 and 3.42 times larger, respectively, than those expected for a compactly folded protein consisting of 109 amino acid residues. A remarkable amount of secondary structure is formed only in the presence of trifluoroethanol at low pH. The finding that a biologically active protein molecule with 109 amino acid residues adopts a random coil conformation under physiological conditions raises the question whether this is a rare or a hitherto-overlooked but widespread phenomenon in the field of macromolecular polypeptides.

The thymus extract Thymex-L potentiates the retinoic acid-induced differentiation of the human myeloid leukemia cell line HL-60.

The human promyelocytic cell line HL-60 can be differentiated with retinoic acid (RA) along the granulocytic pathway. Numerous studies have identified many synergistic combinations of RA with cytostatics, cytokines and other inducers. A combination of RA with the crude thymus extract Thymex-L increased differentiation of HL-60 cells as confirmed by two functional assays and morphology, whereas the extract itself did not show any effect. The functional markers phagocytosis-associated chemiluminescence and nitroblue tetrazolium reduction were more enhanced (up to 4-fold with 1000 micrograms/ml Thymex-L) than morphology. The effect was found over a wide RA concentration range (10(-11) - 10(-6) M) and was dependent on extract concentration. The half-maximal induction of both functional markers was reached at 400 micrograms/ml. To achieve the same effect with the combination in comparison with RA alone, an RA dose reduction of about 100-fold was estimated. The effect was also seen when the cells were pretreated with the thymus extract for two days. The enhancement of RA action by Thymex-L was not correlated with an increase of extracellular or intracellular RA concentration. The active compound in Thymex-L is heat stable and bigger than 5 kDa as confirmed by gelfiltration. The defined thymus peptides thymosin alpha 1, prothymosin alpha 1 and thymopentin were unable to synergistically enhance HL-60 differentiation. These data suggest that the treatment with a thymus extract can increase the sensitivity of HL-60 cells for RA. This may have clinical implications.

Isolation and partial characterization of basic proteinases from stem bromelain.

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substrate L-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine-->serine) and position 20 (asparagine-->glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0:2.0:1.0:2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. The pH optimum of F4 and F5 was between pH 4.0 and 4.5 and for F9 close to neutral pH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM, kcat 0.87 sec-1 and F5 (Km 2.42 mM, kcat 0.68 sec-1), and differed greatly from F9 (Km 0.40 mM, kcat 3.94 sec-1).

Transcription inhibition of SV40 by in vitro DNA methylation.

SV40 DNA was methylated in vitro with prokaryotic or eukaryotic DNA cytosine-5-methyltransferases and the inhibition of transcription by methylation was studied in Xenopus oocytes. Methylation with the prokaryotic HhaI or HpaII methyltransferases did essentially not inhibit transcription of SV40. Methylation with a rat liver methyltransferase led only to minor inhibition of the SV40 early genes, but to a complete shut shut off of the SV40 late genes. Partial methylation showed that methylation of both, the regulatory region and the 5' end of the SV40 late genes, was necessary for the effect on transcription. The TK gene could be inactivated by eukaryotic methylation of either the promoter and the first 50 nucleotides of the gene or the 3' rest of the gene. Insertion of the SV40 enhancer, not containing methylatable CpGs, into the TK upstream region, had no influence on the inhibition of TK gene transcription by methylation.

DNA methylation inhibits transcription by RNA polymerase III of a tRNA gene, but not of a 5S rRNA gene.

Methylation of cytosine in the DNA inhibits the transcription by RNA polymerase II in higher eukaryotes, but has no influence on RNA polymerase I transcription. The effect on RNA polymerase III was unknown, so far. Two polymerase III genes: a type 1 5S rRNA gene and a type 2 tRNA gene were methylated in vitro with a purified eukaryotic DNA methyltransferase (EC2.1.1.37) and their transcription was analyzed in Xenopus oocytes. The 5S rRNA gene, an oocyte 5S rRNA gene from X. laevis which is subject to developmental inactivation, was not affected by methylation. Conversely, transcription of the tRNA gene was 80% inhibited by methylation with the eukaryotic methyltransferase. HhaI and HpaII methylation left its transcription unaffected.

Transcription of HIV1 is inhibited by DNA methylation.

A possible role of DNA methylation as a factor in HIV latency was studied by methylating a HIV1-LTR-CAT plasmid in vitro and measuring its expression after transfection on Vero cells. Methylation with a eukaryotic DNA methylase resulted in a 70% inhibition of chloramphenicol acetyltransferase expression, in the absence as well as in the presence of the HIV1 trans-activator protein TAT in the cell. A similar degree of transcription inhibition was obtained by methylation of the only Hpa II site at position-143 in the HIV1-LTR with the bacterial Hpa II methylase. In contrast to the effect by eukaryotic methylation, the inhibition by Hpa II methylation could be partially reversed by cotransfection of the TAT gene. The reason may lie in an about 40% demethylation at the Hpa II site which was concomitantly observed.