BBA - General Subjects (v.1527, #3)

Effects of lipofectin–antigen complexes on major histocompatibility complex class I-restricted antigen presentation pathway in murine dendritic cells and on dendritic cell maturation by Naoki Okada; Tomomi Saito; Kohei Mori; Yasushige Masunaga; Yoshihiro Fujii; Junko Fujita; Kyoko Fujimoto; Tsuyoshi Nakanishi; Keiichi Tanaka; Shinsaku Nakagawa; Tadanori Mayumi; Takuya Fujita; Akira Yamamoto (97-101).
We previously reported that exogenous antigens complexed with the cationic liposome lipofectin (LF) were efficiently presented via major histocompatibility complex (MHC) class I molecules on pulsed dendritic cells (DCs) in vitro. In the present study, we demonstrated that MHC class I-restricted antigen presentation on DC2.4 cells, a murine immature DC line, treated with LF–antigen complexes was remarkably suppressed through the inhibition of endocytosis, proteasome catalysis, and Golgi transport. We also found that LF did not influence expression of interleukin-12 p40 mRNA, MHC molecules, or co-stimulatory molecules in DC2.4 cells. These findings suggest that an antigen-loading procedure using LF could enhance delivery of exogenous antigens to the classical MHC class I pathway in DCs, but it does not initiate DC maturation.
Keywords: Dendritic cell; Lipofectin; Antigen presentation; Maturation; Immunotherapy;

Legume lectin family, the ‘natural mutants of the quaternary state’, provide insights into the relationship between protein stability and oligomerization by V.R. Srinivas; G.Bhanuprakash Reddy; Nisar Ahmad; Chittoor P. Swaminathan; Nivedita Mitra; Avadhesha Surolia (102-111).
Legume lectins family of proteins, despite having the same ‘jelly roll’ tertiary structural fold at monomeric level, exhibit considerable variation in their quaternary structure arising out of small changes in their sequence. Nevertheless, their folding behavior and stability correlates very well with their patterns of assembly into dimers and tetramers. A conservation of their fold during evolution, its wide distribution in many protein families together with the availability of structural information on them make them interesting as proteins to explore the effect of inter- versus intra-subunit interactions in the stability of multimeric proteins. Additionally, as ‘natural mutants’ of quaternary association, proteins of legume lectin family provide interesting paradigms for studies addressing the effect of subunit oligomerization on the stability, folding and function as well as the evolution of multimeric structures.
Keywords: Legume lectins; Protein stability; DSC; Quaternary structure; Subunit interaction; Evolution of lectin oligomerization;

The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger by Gregg L.F Wallis; Richard J Swift; Robert Atterbury; Susanne Trappe; Ursula Rinas; Frank W Hemming; Marilyn G Wiebe; Anthony P.J Trinci; John F Peberdy (112-122).
The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied. We have shown that a change in the pH from 4 to 5.4 during continuous cultivation of the A. niger B1 strain instigates or accelerates the spontaneous generation of a morphological mutant (LB). This mutant strain produced approx. 50% less extracellular protein and GAM during both chemostat and batch cultivation compared to another strain with parental-type morphology (PS). The intracellular levels of GAM were also lower in the LB strain. In addition, cultivation of the original parent B1 strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold drop in GAM production and a 5-fold drop in extracellular protein compared to that obtained at pH 4. Glycosylation analysis of the glucoamylases purified from shake-flask cultivation showed that both principal forms of GAM secreted by the LB strain possessed enhanced galactosylation (2-fold), compared to those of the PS. Four diagnostic methods (immunostaining, mild methanolysis, mild acid hydrolysis and β-galactofuranosidase digestion) provided evidence that the majority of this galactose was of the furanoic conformation. The GAMs produced during batch-pulse cultivation at pH 5.5 similarly showed an approx. 2-fold increase in galactofuranosylation compared to pH 4. Interestingly, in both cases the increased galactofuranosylation appears primarily restricted to the O-linked glycan component. Ambient pH therefore regulates both GAM production and influences its glycosylation.
Keywords: pH; Glucoamylase; Galactofuranose; Aspergillus niger;

The preparation of magnetic proteinaceous microspheres using the sonochemical method by S Avivi (Levi); I Felner; I Novik; A Gedanken (123-129).
Using high-intensity ultrasound, we have developed a method for the synthesis of magnetic microspheres. The microspheres are composed of iron oxide-filled and coated globular bovine serum albumin (BSA). The magnetic microspheres are prepared from BSA and iron pentacarbonyl, or from BSA and iron acetate. Transmission electron microscopy and scanning electron microscopy show spherical particles. The particle size distributions are gaussian, with a mean diameter of a few micrometers. Using chemical analysis, it was found that the total percentage of iron oxide in the microspheres is between 39% and 42%. Mössbauer measurements were also performed.
Keywords: Proteinaceous microspheres; Sonochemistry; Amorphous iron oxide; Nanoparticles; Magnetic nanoparticles;

Elongation factor 1β is an actin-binding protein by Ruth Furukawa; Tim M Jinks; Tomer Tishgarten; Mark Mazzawi; Donald R Morris; Marcus Fechheimer (130-140).
A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1β. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1β. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1β. Direct interaction of eEF1β with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1β, recombinant preparations of Dictyostelium eEF1β expressed in Escherichia coli, and the intact eEF1βγ complex purified from wheat germ. Localization of eEF1β in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1β may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.
Keywords: Elongation factor 1; Actin binding protein; Cytoskeleton; Protein synthesis; Dictyostelium;

Dicynthaurin: an antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium by In Hee Lee; Young Shin Lee; Chong Han Kim; Chung Ryul Kim; Teresa Hong; Lorenzo Menzel; Lee Ming Boo; Jan Pohl; Mark A. Sherman; Alan Waring; Robert I. Lehrer (141-148).
We isolated a novel antimicrobial peptide, dicynthaurin, from hemocytes of a tunicate, Halocynthia aurantium. The native peptide had a mass of approximately 6.2 kDa and was composed of two 30-residue monomers without sequence homology to any previously identified peptides (ILQKAVLDCLKAAGSSLSKAAITAIYNKIT). Most cynthaurin molecules were C-terminally amidated and were linked covalently by a single cystine disulfide bond. When performed in membrane-mimetic environments, circular dichroism studies of dicynthaurin revealed largely α-helical conformations. Dicynthaurin’s broad-spectrum activity encompassed Gram-positive (Micrococcus luteus, Staphylococcus aureus, Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), but not Candida albicans, a fungus. Although dicynthaurin was purified from a marine invertebrate, its antimicrobial activity was optimal at NaCl concentrations below 100 mM. This suggests that the antimicrobial actions of this molecule may take place intracellularly (e.g., within a phagosome) rather than extracellularly.
Keywords: Antimicrobial; α-Helical; Dimer; Hemocyte; Peptide; Tunicate;

Cell age-related monovalent cations content and density changes in stored human erythrocytes by Giampaolo Minetti; Annarita Ciana; Antonella Profumo; Manuela Zappa; Cristina Vercellati; Alberto Zanella; Arduino Arduini; Augusta Brovelli (149-155).
Conversion of erythrocyte membrane protein 4.1b to 4.1a occurs through a non-enzymatic deamidation reaction in most mammalian erythrocytes, with an in vivo half-life of approximately 41 days, making the 4.1a/4.1b ratio a useful index of red cell age [Inaba and Maede, Biochim. Biophys. Acta 944 (1988) 256–264]. Normal human erythrocytes distribute into subpopulations of increasing cell density and cell age when centrifuged in polyarabinogalactan density gradients. We have observed that, when erythrocytes were stored at 4°C under standard blood bank conditions, the deamidation was virtually undetectable, as cells maintained the 4.1a/4.1b ratio they displayed at the onset of storage. By measuring the 4.1a/4.1b values in subpopulations of cells of different density at various time points during storage, a modification of the normal ‘cell age/cell density’ relationship was observed, as erythrocytes were affected by changes in cell volume in an age-dependent manner. This may stem from a different impact of storage on the imbalance of monovalent cations, Na+ and K+, in young and old erythrocytes, related to their different complement of cation transporters.
Keywords: Human erythrocyte; Protein 4.1a; Protein 4.1b; Monovalent cation; Storage; Cell age;

In situ observation of the generation of isothiocyanates from sinigrin in horseradish and wasabi by Eileen Y Yu; Ingrid J Pickering; Graham N George; Roger C Prince (156-160).
Sulfur K-edge X-ray absorption spectroscopy has been used to determine the chemical identity of the sulfur-containing species in horseradish (Armoracia lapthifolia) and wasabi (Wasabia japonica) in situ, before and after cell disruption. The major sulfur-containing species in the intact root is sinigrin (1-thio-β-d-glucopyranose 1-N-(sulfoxy)-3-buteneimidate) and related congeners. Disrupting the cells by applying local pressure allowed the conversion of the sulfur moieties in sinigrin to isothiocyanates and sulfate in approximately equimolar amounts. In contrast to previous suggestions, no detectable thiocyanates were formed, but an unusual thio intermediate may have been identified for the first time.
Keywords: X-ray absorption spectroscopy; Isothiocyanate; Sinigrin; Myrosinase;

It is proposed that bile acids (deoxycholic acid), the K vitamins, iron(II) complexes and oxygen interact to induce an oncogenic effect in the colon by the generation of free radicals. In the relatively low oxidising/reducing conditions of the colonic lumen the K vitamins exist in the reduced form; however, if absorbed into the mucosa they have the capacity to be chemically oxidised and to enter into a redox cycle yielding oxygen radicals. The semiquinone radical of K1 (phylloquinone) has been stabilised in bile acid mixed micelles and investigated by electron paramagnetic resonance spectroscopy and quantum chemical calculations. The estimated half-life of the radical was about 30 min which confirms a remarkably high stability in aqueous micellar solution. A model is presented in which the reduced K vitamins may initiate superoxide radical, O−⋅ 2 generation leading to Fe(II) mediated Fenton reactions in the stem colon cells.
Keywords: Colon cancer; Free radical; Vitamin K; Electron paramagnetic resonance spectroscopy; Superoxide;

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rabbits by Antonella Bertazzo; Eugenio Ragazzi; Monica Biasiolo; Carlo V.L Costa; Graziella Allegri (167-175).
The following enzyme activities of the tryptophan-nicotinic acid pathway were studied in male New Zealand rabbits: liver tryptophan 2,3-dioxygenase, intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Intestine superoxide dismutase and serum tryptophan were also determined. Liver tryptophan 2,3-dioxygenase exists only as holoenzyme, but intestine indole 2,3-dioxygenase is very active and can be considered the key enzyme which determines how much tryptophan enters the kynurenine pathway also under physiological conditions. The elevated activity of indole 2,3-dioxygenase in the rabbit intestine could be related to the low activity of superoxide dismutase found in intestine. Kynurenine 3-monooxygenase appeared more active than kynurenine-oxoglutarate transaminase and kynureninase, suggesting that perhaps a major portion of kynurenine available from tryptophan may be metabolized to give 3-hydroxyanthranilic acid, the precursor of nicotinic acid. In fact, 3-hydroxyanthranilate 3,4-dioxygenase is much more active than the other previous enzymes of the kynurenine pathway. In the rabbit liver 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase show similar activities, but in the kidney 3-hydroxyanthranilate 3,4-dioxygenase activity is almost double. These data suggest that in rabbit tryptophan is mainly metabolized along the kynurenine pathway. Therefore, the rabbit can also be a suitable model for studying tryptophan metabolism in pathological conditions.
Keywords: Tryptophan metabolism; Kynurenine pathway; Enzyme activity; Rabbit;

Contents Vol. 1527 (178-179).