A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A

An alpha-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4(E249K), Pdcd4(D253A), Pdcd4(D414K), Pdcd4(D418A), Pdcd4(E249K,D414K), and Pdcd4(D253A,D418A)) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4(D253A) and Pdcd4(D253A,D418A) do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4(D253A), Pdcd4(D418A), or Pdcd4(D235A,D418A). Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.     

Binary mixtures of (N-phosphonomethyl)-glycine with new aminophosphonates

The potential biological activity of binary mixtures of some new organophosphorous compounds, aminoalkane- and aminofluorenephosphonates, with (N-phosphonomethyl)-glycine (glyphosate, PMG) was studied. The inhibition of growth of wheat (Triticum aestivum) induced by individual compounds and their equimolar mixtures with PMG was a measure of that activity. The experiments were expected to show if the new compounds exhibited good biological activity to be used for agrochemical applications and if this activity can be improved when they are used in mixtures with glyphosate which is the active component of the well-known herbicide Roundup. The results obtained show that aminofluorenephosphonates inhibited wheat growth when used in micromolar concentrations. Thus, their efficiency can be compared to that of PMG. The efficiency of aminoalkanephosphonates was one order of magnitude weaker. The measure of the efficiency was the effective concentration inhibiting wheat growth by 50% (EC50). The most demanded interaction, i.e., a synergistic was observed for only one of binary mixtures of the compounds studied with PMG. Mostly they showed antagonistic or strong antagonistic interactions. Some of them were of the additive type. Such results exclude the possibility of potential use of all the compounds studied in binary mixtures with phosphonomethylglycine, especially as the mentioned synergistic interaction found was rather weak. The influence of structural features of anminophosphonates on the results obtained is discussed.     

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.     

Further development of local IL-2 therapy of cancer: multiple versus single IL-2 treatment of transplanted murine colon carcinoma

We have compared the effect of one and up to four local IL-2 treatments of transplanted MC38 colon carcinoma. A single IL-2 treatment prolonged the survival time (p=0.015), but no cure was obtained. One local IL-2 treatment inhibited tumor growth for about 1 week. After the start of tumor regrowth, a further IL-2 injection was given. After four IL-2 injections 6 out of 13 mice were cured. Histological studies show that IL-2 induced a local vascular leakage syndrome leading to massive peritumoral edema and subsequent necrosis of tumor tissue. IL-2 also attracted infiltrating cells, mainly macrophages. Subsequent IL-2 injections led to complete tumor regression. We believe that the combination of necrotic tumor debris and the IL-2–induced macrophage reaction enhanced a tumor-specific immune response. This local IL-2 application was not toxic.     

Soluble intercellular adhesion molecule-1 (sICAM-1): an overview

Soluble intercellular adhesion molecule-1 (sICAM-1) represents a circulating form of ICAM-1 that is constitutively expressed or is inducible on the cell surface of different cell lines. It serves as a counter-receptor for the lymphocyte function-associated antigen (LFA-1). Interaction between ICAM-1, present on endothelial cells, and LFA-1 facilitates leukocyte adhesion and migration across the endothelium. ICAM-1 and its circulating form have been implicated in the development of any number of diseases.     

Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays

We present a genotyping method for simultaneously scoring 116,204 SNPs using oligonucleotide arrays. At call rates >99%, reproducibility is >99.97% and accuracy, as measured by inheritance in trios and concordance with the HapMap Project, is >99.7%. Average intermarker distance is 23.6 kb, and 92% of the genome is within 100 kb of a SNP marker. Average heterozygosity is 0.30, with 105,511 SNPs having minor allele frequencies >5%.     

Oral feeding with ethinyl estradiol suppresses and treats experimental autoimmune encephalomyelitis in SJL mice and inhibits the recruitment of inflammatory cells into the central nervous system

There is much interest in the possible ameliorating effects of estrogen on various autoimmune diseases. We previously established the protective effects of 17 beta-estradiol (E2) on experimental autoimmune encephalomyelitis (EAE). In the current study we investigated the effectiveness of oral treatment with ethinyl estradiol (EE) on EAE and the mechanisms involved. Ethinyl estradiol is a semisynthetic estrogen compound found in birth control pills, and its chemical structure allows this compound to retain activity when given orally. We found that oral EE, like E2, drastically suppressed EAE induced by proteolipid protein 139-151 peptide when given at initiation of EAE. However, unlike E2, EE reduced clinical severity when given after the onset of clinical signs. Treatment with EE significantly decreased the secretion of proinflammatory cytokines (IFN-gamma, TNF-alpha, and IL-6) by activated T cells as well as the expression of a key matrix metalloproteinase, disease-mediating chemokines/receptors, and IgG2a levels, but increased the expression of TGF-beta 3 in the CNS. The absence of infiltrating lymphocytes together with the suppression of cytokines, matrix metalloproteinase, and chemokines/receptors suggests that EE, like E2, protects mice from EAE by inhibiting the recruitment of T cells and macrophages into the CNS. These results suggest that oral ethinyl estradiol might be a successful candidate as therapy for multiple sclerosis.     

Novel bacterial polar lipids containing ether-linked alkyl chains,the structures and biological properties of the four major glycolipids from Propionibacterium propionicum PCM 2431 (ATCC 14157T)

Propionibacterium propionicum belongs to the "acnes group" of propionibacteria, which is currently considered as clinically important because of its growing potential in infections, in particular with those connected with immune system dysfunctions. Propionibacteria are thought to be actinomycete-like microorganisms and may still cause diagnostic difficulties. The chloroform-methanol extracts of the cell mass of P. propionicum (type strain) gave in TLC analysis the characteristic glycolipid profile containing four major glycolipids, labeled G(1) through G(4). These polar lipids were found to be useful chemotaxonomic markers to differentiate P. propionicum from other cutaneous propionibacteria, in particular from strains of the acnes group. Glycolipids G(1)-G(4) were isolated and purified using gel-permeation chromatography, TLC, and high performance liquid chromatography, and their structures were elucidated by compositional and methylation analyses, specific chemical degradations, MALDI-TOF mass spectrometry, and (1)H NMR and (13)C NMR spectroscopy, including HMBC, TOCSY, HMQC, and NOESY experiments. Glycolipids G(2) and G(3) possess as backbone alpha-d-Glcp-(1 --> 3)-alpha-d-Glcp-(1 --> 1)-Gro (Gro, glycerol), in which position O-2 of the glycerol residue is acylated by a fatty acid (mainly C(15):0) while O-3 is substituted by an alkyl ether chain. In glycolipid G(3), an additional fatty acyl chain was linked to O-6 of the terminal glucose residue. Glycolipid G(4) was structurally related to G(2) but devoid of one glucose residue. Glycolipid G(1) was isolated in small amounts, and its structure was therefore deduced from MALDI-TOF-MS experiments alone, which revealed that it possessed the structure of G(2) but was lacking one fatty acid residue. In studies on the biological properties of P. propionicum glycolipids, the anti-P. propionicum rabbit antisera reacted in dot enzyme-immunoblotting test with G(2) and G(3). Glycolipid G(3) was able to induce the delayed type of hypersensitivity. The results indicated that these novel ether linkage-containing polar glycolipids are immunogenic and possibly active in hypersensitivity, and thus, in pathogenesis.     

A cross-platform public domain PC image-analysis program for the comet assay

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.