Chemical cross-linking, mass spectrometry, and in silico modeling of proteasomal 20S core particles of the haloarchaeon Haloferax volcanii

A fast and accurate method is reported to generate distance constraints between juxtaposited amino acids and to validate molecular models of halophilic protein complexes. Proteasomal 20S core particles (CPs) from the haloarchaeon Haloferax volcanii were used to investigate the quaternary structure of halophilic proteins based on their symmetrical, yet distinct subunit composition. Proteasomal CPs are cylindrical barrel-like structures of four-stacked homoheptameric rings of α- and β-type subunits organized in α(7)β(7) β(7)α(7) stoichiometry. The CPs of H. volcanii are formed from a single type of β subunit associated with α1 and/or α2 subunits. Tandem affinity chromatography and new genetic constructs were used to separately isolate α1(7)β(7)β(7)α1(7) and α2(7)β(7)β(7)α2(7) CPs from H. volcanii. Chemically cross-linked peptides of the H. volcanii CPs were analyzed by high-performance mass spectrometry and an open modification search strategy to first generate and then to interpret the resulting tandem mass spectrometric data. Distance constraints obtained by chemical cross-linking mass spectrometry, together with the available structural data of nonhalophilic CPs, facilitated the selection of accurate models of H. volcanii proteasomal CPs composed of α1-, α2-, and β-homoheptameric rings from several different possible structures from Protein Data Bank.     

A Comparative Study of In Vitro Cytotoxic,Antioxidant, and Antimicrobial Activity of Pt(II), Zn(II), Cu(II), and Co(III) Complexes with N‐heteroaromatic Schiff Base (E)‐2‐[N′‐(1‐pyridin‐2‐yl‐ethylidene)hydrazino]acetate

In search for novel biologically active metal based compounds, an evaluation of in vitro cytotoxic, antioxidant, and antimicrobial activity of new Pt(II) complex and its Zn(II), Cu(II), and Co(III) analogues, with NNO tridentately coordinated N‐heteroaromatic Schiff base ligand (E)‐2‐[N′‐(1‐pyridin‐2‐yl‐ethylidene)hydrazino]acetate, was performed. Investigation of antioxidative properties showed that all of the compounds have strong radical scavenging potencies. The Zn(II) complex showed potent inhibition of DNA cleavage by hydroxyl radical. A cytotoxic action of investigated compounds was evaluated on cultures of human promyelocitic leukaemia (HL‐60), human glioma (U251), rat glioma (C6), and mouse melanoma (B16) cell lines. It was shown that binuclear pentacoordinated Zn(II) complex possesses a strong dose‐dependent cytotoxic activity, of the same order of magnitude as cisplatin on B16, C6, and U251 cells. Furthermore, Zn(II) complex causes oxidative stress‐induced apoptotic death of HL‐60 leukemic cells, associated with caspase activation, phosphatidylserine externalization, and DNA fragmentation.     

Synthesis and anti-enterovirus activity of new analogues of MDL-860

A series of twelve novel compounds, analogues of antiviral agent MDL-860 were synthesized and their antiviral activity was evaluated in vitro against enteroviruses poliovirus 1 (PV1), Coxsackieviruses B1 (CVB1) and Coxsackieviruses B3 (CVB3). Compounds 14, 24 and 25 manifested strong antiviral effects against CVB1 and PV1 (SI values of 405 and 118 for CVB1 and PV1 respectively). In contrast to the wide anti-enteroviral activity of MDL-860, these three compounds were inactive against CVB3. Compounds 14, 24 and 25 along with MDL-860 were tested in vivo in mice infected with CVB1. Marked protective effects of compounds 14 and 24 were established, PI values of 50% and 33.3%, respectively. In addition, almost all of the tested compounds manifested very low toxicity.     

Synthesis and reactivity of the monosaccharide esters of amino acids as models of teichoic acid fragment

The increasing prevalence of sepsis from Gram-positive bacterial pathogens necessitates further evaluation of the basic assumptions about the molecular pathogenesis of septic shock. Since diverse physiological functions of Gram-positive bacteria are controlled by the degree of esterification of teichoic acids with D-alanine, we examined the reactivity of monosaccharide esters in which anomerically free or protected D-glucose is linked through its C-6 hydroxy group to either phenylalanyl or tyrosyl residues as models for teichoic acid fragment. We show that the attached sugar moiety induces activation of the amino acid residue. Due to the enhanced reactivity of the NH₂ group in the monosaccharide esters studied, the formation of products generated by intramolecular and intermolecular glycation reactions is accelerated resulting in heterogeneous mixture of compounds. These findings suggest that, if similar adducts are formed by glycation of D-alanine in teichoic acid of Gram-positive bacteria, they should be examined as potential bioactive ligands or chemical message for infection.     

In vitro and in vivo toxicity evaluation of the freshwater cyanobacterium Heteroleiblenia kuetzingii

Cyanobacteria are prokaryotic organisms characterized by their ability to produce secondary metabolites with different biological activities. The aim of this work was to evaluate the in vitro and in vivo toxicity of the cosmopolitan freshwater cyanobacterium H. kuetzingii. An extract from H. kuetzingii and cyanobacterial growth media were assessed for presence of intracellular and extracellular toxins by in vitro tests using primary cell cultures from mouse kidney and fibroblasts, cell lines A549 and 3T3, a fish cell line RTgill-W1 as well as by a traditional in vivo mouse bioassay. The presence of toxicity was compared with the ELISA and HPLC data for corresponding cyanotoxins. In vitro tests showed pronounced cytotoxicity of the cyanobacterium extract and growth medium in which H. kuetzingii released potential extracellular toxic compounds as the mammalian cells were significantly more sensitive to exposure compared to the fish cells. Histopathological analyses of the liver and kidneys of treated mice showed pathological changes such as leukocyte infiltration and necrosis, changes in the proximal and distal convoluted tubules, lack of differentiation of Bowman’s space, enlarged Bowman’s capsules and massive hemorrhages. ELISA and HPLC analyses confirmed the presence of saxitoxins and microcystins at low concentrations. In addition, the histological analyses suggest that H. kuetzingii produces other, yet unknown toxic metabolites. Monitoring efforts are therefore required to evaluate the potential hazard for the freshwater aquatic systems and possible public health implications associated with this cyanobacterium.     

Non-enzymatic glycation of elastin

Non-enzymatic glycation of proteins is one of the key mechanisms in the pathogenesis of diabetic complications and may be significant in the age-related changes of tissues. We investigated thein vitro glycation of human aortic -elastin, and chose and adapted methods for evaluating the degree and kinetics of glycation. -Elastin was prepared from thoracic aortas of young accident victims and glycated by incubating with different glucose concentrations (25, 50, 75 and 100 mmol/l) in 0.2 M phosphate buffer, pH 7.8 for 30 days, at 37°C. The degree of glycation was measured by three colorimetric methods,i.e. Nitroblue tetrazolium, 2-thiobarbituric acid and hydrazine; by aminophenyl-boronate affinity chromatography which determines Amadori products; and by a fluorescence method which determines advanced glycosylation end products. The highest degree of glycation was found on day 3 after the beginning of incubation. Fluorescence, as an index of advanced glycation, consistently increased from days 5 to 24. Investigation of the properties of glycated elastin may help in understanding the importance of this long-lived protein for the age-related changes in tissues and for diabetic complications.     

Role of nitrogen sources and metal ions in urease synthesis byMicrococcus varians

Urease (urea amidohydrolase E.C.3.5.1.5) synthesis inMicrococcus varians U-9 was not affected by nitrogen source (peptone or glutamic acid) or its concentration: but depended on the ratio of peptone and urea in culture medium. WhenM. varians grew in culture medium with peptone at or above 0.48g/l and NH₄Cl as an additional nitrogen source, two maxima of urease synthesis occurred; one in the exponential growth phase and the second in the stationary growth phase. Though this bacterium could not utilize either urea or ammonia as the sole nitrogen source, urea caused only one maximum of urease synthesis to occur and shifted the maximum into late exponential phase, suggesting that urea acts as a regulatory factor in urease synthesis. Synthesis of urease was not induced either by urea or by nitrogen starvation and was not repressed by ammonia or by excess of complex nitrogen source. NI²⁺ (up to 0.1 mM) stimulated urease synthesis but decreased bacterial growth, while Co²⁺ only affected bacterial growth and at 0.1 mM Inhibited the growth.

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Résumé La synthèse d'uréase (urée-amidohydrolase E.C.3.5.1.5) chezMicrococcus varians n'est pas affectée par la source d'azote (peptone ou acide glutamique), ni par leurs concentrations, mais dépend du rapport de la peptone à l'urée dans le milieu de culture. QuandM. varians croît dans un milieu de culture contenant la peptone à la concentration égale ou supérieure à 0.48 g/l et le NH₄Cl comme source additionnelle d'azote, on observe deux maximum de synthèse d'uréase: la première dans la phase exponentielle de croissance, et la seconde dans la phase stationnaire de croissance. Blen que cette bactérie ne puisse utilliser ni l'urée ni l'ammoniac comme seule source d'azote, l'urée provoque un seul des maximum de synthèse d'uréase et déplace ce maximum vers la partie ultime de la phase exponentielle, suggérant que l'urée agit comme facteur de régulation dans la synthèse d'uréase. La synthèse d'uréase n'est induite ni par l'urée ni par la starvation d'azote et n'est réprimée ni par l'ammoniac ni par un excès de la source d'azote complexe. Le Ni²⁺ (jusqu'à 0.1 mM) stimule la synthèse d'uréase, mais diminue la croissance bactérienne, tandis que le Co²⁺ n'affecte que la croissance bactérienne et à 0.1 mM inhibe la croissance.

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This work was carried out at the Faculty of Food Technology and Biotechnology, University of Zagreb. Department of Biochemical Engineering, 41000 Zagreb, Pierottijeva 6/IV, Croatia, Yugoslavia.     

Cataract surgery and intraocular lens implantation in children with juvenile rheumatoid arthritis associated uveitis

Clinical records of 6 children (7 eyes) with juvenile rheumatoid arthritis (JRA) who underwent cataract surgery with IOL implantation between January 1998 and December 2002 were reviewed. The median age at the time of cataract surgery was 8 years (range 5-14 years). The median follow up was 48 months (range 26 to 60 months). Five of six children (6 eyes) were on systemic immunosuppressive or anti-inflammatory therapy. Glaucoma was present in three eyes before surgery, and all three eyes underwent combined cataract surgery and trabeculectomy with mitomycin C. A final best corrected visual acuity of 0.5 or better was achieved in all eyes Postoperative complications included posterior capsule opacification (n = 5), glaucoma (n = 1), and cystoid macular edema (n = 1). Intraocular lens implantation in children with control of preoperative and postoperative ocular inflammation could lead to favorable visual results.