Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.     

Vitality diagnostics and preventive medicine for the well-being of people at advanced age

This paper emphasises the importance of vitality diagnostics in relation to healthy ageing and prevention of age-associated diseases. Ageing, reducing the reserve capacity, decreases the adaptability of various systems and increases the risk of functional disorders. The early recognition and treatment of functional disorders in vitality diagnostics laboratories provides an opportunity to prevent or delay the onset of degenerative diseases related to advanced age.     

Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.     

Combined millimeter wave and cyclophosphamide therapy of an experimental murine melanoma

The objective of the present studies was to investigate whether millimeter wave (MMW) therapy can increase the efficacy of cyclophosphamide (CPA), a commonly used anti-cancer drug. The effect of combined MMW-CPA treatment on melanoma growth was compared to CPA treatment alone in a murine model. MMWs were produced with a Russian made YAV-1 generator. The device produced 42.2 +/- 0.2 GHz modulated wave radiation through a 10 x 20 mm rectangular output horn. The animals, SKH-1 hairless female mice, were irradiated on the nasal area. Peak SAR and incident power density were measured as 730 +/- 100 W/kg and 36.5 +/- 5 mW/cm2, respectively. The maximum skin surface temperature elevation measured at the end of 30 min irradiation was 1.5 degrees C. B16F10 melanoma cells (0.2 x 10(6)) were implanted subcutaneously into the left flank of mice on day 1 of the experiment. On days 4-8, CPA was administered intraperitoneally (30 mg/kg/day). MMW irradiation was applied concurrently with, prior to or following CPA administration. A significant reduction (P < .05) in tumor growth was observed with CPA treatment, but MMW irradiation did not provide additional therapeutic benefit as compared to CPA alone. Similar results were obtained when MMW irradiation was applied both prior to and following CPA treatment.     

Baculovirus expression system for heterologous multiprotein complexes

The discovery of large multiprotein complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant multiprotein production and multigene transfer applications.     

Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.     

Elevated levels of matrix metalloprotein-3 in patients with coronary aneurysm: A case control study

BACKGROUND: Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of arterial aneurysms through increased proteolysis of extracellular matrix proteins. Increased proteolysis due to elevated matrix degrading enzyme activity in the arterial wall may act as a susceptibility factor for the development of coronary aneurysms. The aim of this study was to investigate the association between MMPs and presence of coronary aneurysms. METHODS: Thirty patients with aneurysmal coronary artery disease and stable angina were enrolled into study (Group 1). Fourteen coronary artery disease patients with stable angina were selected as control group (Group 2). MMP-1, MMP-3 and C-reactive protein (CRP) were measured in peripheral venous blood and matched between the groups. RESULTS: Serum MMP-3 level was higher in patients with aneurismal coronary artery disease compared to the control group (20.23 +/- 14.68 vs 11.45 +/- 6.55 ng/ml, p = 0.039). Serum MMP-1 (13.63 +/- 7.73 vs 12.15 +/- 6.27 ng/ml, p = 0.52) and CRP levels (4.78 +/- 1.47 vs 4.05 +/- 1.53 mg/l, p = 0.13) were not significantly different between the groups. CONCLUSION: MMPs can cause arterial wall destruction. MMP-3 may play role in the pathogenesis of coronary aneurysm development through increased proteolysis of extracellular matrix proteins.     

Physical mapping of the bovine immunoglobulin heavy chain constant region gene locus

Bovine antibodies have recently attracted increasing attention, as they have been shown to exhibit prophylactic and therapeutic properties in selected infectious diseases in humans. In the present study, we have isolated bacterial artificial chromosomes and cosmid clones containing the bovine JH, mu, delta, gamma 1, gamma 2, gamma 3, epsilon, and alpha genes, which allowed us to make a contig of the genes within the bovine IGHC locus. The genes are arranged in a 5'-JH-7 kb-mu-5 kb-delta-33 kb-gamma 3-20 kb-gamma 1-34 kb-gamma 2-20 kb-epsilon- 13 kb-alpha-3' order, spanning approximately 150 kb DNA. Examination of the bovine germline JH locus revealed six JH segments, two of which, JH1 and JH2, were shown to be functional although there was a strong preference for expression of the former. Sequence alignment of the bovine 5' E mu enhancer core region with those of other mammals, demonstrated an absence of the mu E3 motif and a shortened spacer between the mu A and mu B sites within the bovine E mu enhancer core region. Furthermore, the essential sequence element for class switching, switch mu, spanning approximately 3-kb repetitive sequence and abundant in the switch region motifs CTGGG (187 repeats) and CTGAG (127 repeats), was identified immediately upstream of the mu gene. A further sequence comparison revealed that the bovine IGHC genes display an extensive polymorphism leading to expression of multiple antibody allotypes.