Effect of nitrogen source and concentration to produce proteins in mass cultures of the microalgae <i>Chaetoceros muelleri</i>

Proteins are one of the major metabolites in biomass from microalgae that constitute the diet of marine organisms grown in aquaculture, and are essential for their growth. The quantity of this component is influenced by nutrients, temperature and light intensity, among others. We examined the growth, biomass production and protein of Chaetoceros muelleri with two sources of nitrogen (nitrate and urea) at three concentrations, using the medium f/2 (0.88 mol/L) (nitrates) as control. The treatments were the medium 2f (3.53 mol/L) and 4f (7.05 mol/L) with NO_3^-, and the medium f/2 (0.88 mol/L), 2f (3.53mol/L) and 4f (7.05 mol/L) with urea. In general, the productive parameters were greater using urea than nitrate in the media. Higher cell concentrations (2.83 x 10⁶ cell/mL), average and cumulative growth rates (1.50 div/day and 6.01 divisions), dry weight (0.0044 g/L), and proportion of proteins (23.74%) were found when urea was used as the N source. However, most of the bands on the electrophoretic profile were present in the mediums with NO_3^- (~6.5 to 90 kDa).     

Constitutive expression of IL-22 in the human intervertebral disc and its reduction by exposure to pro-inflammatory cytokines in vitro

The importance of cytokines in disc degeneration is well recognized. Little is known about IL-22 expression in the human intervertebral disc. We investigated IL-22 immuno-localization in disc tissue, and molecular expression and production of IL-22 by annulus cells cultured in three-dimensional (3D) culture. We examined human disc tissue using immunohistochemistry and we cultured isolated annulus cells in 3D to analyze IL-22 expression and production, and its receptor, IL-22R, in conditioned media. Ingenuity pathway analysis (IPA) also was used to identify significant gene expression networks within the molecular data. IL-22 and IL-22R were immunolocalized in many cells in the human outer and inner annulus; fewer cells exhibited localization in the nucleus. Three-dimensional culture of annulus cells demonstrated production of IL-22 in conditioned media; exposure to IL-1ß or TNF-α significantly reduced IL-22 levels. Significant decreases also were identified in conditioned media assayed for IL-22R in TNF-α treated cells. IPA analysis showed that IL-22 ranked among the top canonical pathways. We found constitutive expression and production of IL-22 and IL-22R in the disc, which expands our understanding of the effect of pro-inflammatory cytokines on IL-22 expression and production. Three-dimensional cultured annulus cells exposed to IL-1ß or TNF produced significantly lower levels of IL-22 into their conditioned media compared to levels produced by control cells. Our findings have clinical relevance because of the elevated pro-inflammatory milieu within the degenerating human disc.     

Description of Euroleptochromus gen.n. (Coleoptera,Staphylinidae, Scydmaeninae) from Baltic amber,with discussion of biogeography and mouthpart evolution within Clidicini

A new extinct species of the ant‐like stone beetle supertribe Mastigitae, Euroleptochromus sabathi gen. & sp.n. is described from Eocene Baltic amber. A phylogenetic analysis of Clidicini, with representatives of Leptomastacini and Mastigini as out‐group taxa, provided strong support for a sister‐group relationship between the Neotropical Leptochromus and the new genus. The monophyly of Clidicini is questioned because of an alternative placement of Nearctic Papusus as a sister taxon to Leptomastacini + [Clidicus + (Palaeoleptochromus + (Euroleptochromus + Leptochromus))]. A dispersal‐vicariance analysis provided three alternative scenarios for the evolution of Mastigitae; with Laurasia as the ancestral area of the supertribe, major branching events occurring within either Eurasia or Laurentia and two trans‐Beringia dispersals in Late Cretaceous and Eocene. Euroleptochromus, Palaeoleptochromus and Leptochromus share highly derived structures on postgenae and maxillary palps, probably as part of a specialised feeding or prey capture mechanism. The formation of these modifications in Clidicini is demonstrated to involve a process (traced back to the Campanian, 79 Ma) of elongation and narrowing of maxillary palps and forming a cuticular setal projection from a broadened insertion site of sensory setae.     

A transient kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase

Spectral and kinetic features of the redox intermediates of human recombinant unprocessed monomeric myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary cell line, were studied by the multi-mixing stopped-flow technique. Both the ferric protein and compounds I and II showed essentially the same kinetic behavior as the mature dimeric protein (MPO) isolated from polymorphonuclear leukocytes. Firstly, hydrogen peroxide mediated both oxidation of ferric recMPO to compound I (1.9 x 10(7) M(-1) s(-1), pH 7 and 15 degrees C) and reduction of compound I to compound II (3.0 x 10(4) M(-1) s(-1), pH 7 and 15 degrees C). With chloride, bromide, iodide and thiocyanate compound I was reduced back to the ferric enzyme (3.6 x 10(4) M(-1) s(-1), 1.4 x 10(6) M(-1) s(-1), 1.4 x 10(7) M(-1) s(-1) and 1.4 x 10(7) M(-1) s(-1), respectively), whereas the endogenous one-electron donor ascorbate mediated transformation of compound I to compound II (2.3 x 10(5) M(-1) s(-1)) and of compound II back to the resting enzyme (5.0 x 10(3) M(-1) s(-1)). Comparing the data of this study with those known from the mature enzyme strongly suggests that the processing of the precursor enzyme (recMPO) into the mature form occurs without structural changes at the active site and that the subunits in the mature dimeric enzyme work independently.     

The consequence of passive administration of an anti-human immunodeficiency virus type 1 neutralizing monoclonal antibody before challenge of chimpanzees with a primary virus isolate.

The anti-gp41 virus neutralizing monoclonal antibody 2F5 was infused into chimpanzees, which were then given an intravenous challenge with a primary human immunodeficiency virus type I (HIV-1) isolate. In two control animals, the infection was established immediately, as evidenced by positive cell-associated DNA PCR and serum RNA PCR tests within 1 week, seroconversion by 4 weeks, and development of lymphadenopathy in this acute phase. Serum RNA PCR tests were negative in one of the two antibody-infused animals until week 8 and in the other antibody-infused animal until week 12; both animals seroconverted at week 14. The peak of measurable virus-specific serum RNA was delayed until week 16 in one antibody-infused animal. Virus-specific RNA in the other animal did not reach levels comparable to those in the other animals through 1 year of follow-up studies. Virus was isolated from the week 16 blood sample from one infused animal. Virus was not isolated from peripheral blood of the second animal but was isolated from lymph node cells taken at week 36. The infection of untreated chimpanzees with this primary isolate appears robust. Use of this isolate should widen the scope of possible experiments in the chimpanzee model. This antibody infusion study indicates that neutralizing antibody, when present at the time of challenge, affects the timing and level of infection and remains influential after it can no longer be detected in the peripheral circulation. It is possible that preexisting, neutralizing antibodies (passively administered or actively elicited) affect the course of acute-phase virus replication in humans. It remains to be established whether these immunologically mediated early effects will influence the course of HIV-1 disease.     

Glu375Gln and Asp225Val mutants: about the nature of the covalent linkages between heme group and apo-Protein in bovine lactoperoxidase

In analogy with studies previously reported for myeloperoxidase (Kooter, I. M.; Moguilevsky, N.; Bollen, A.; Van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. J. Biol. Chem. 1999, 274, 26794), we examined for bovine lactoperoxidase the effect of mutation of Asp225 and Glu375, the residues thought to be responsible for the covalent binding of the heme group to the apoprotein. Starting from the plasmid encoding rbLPO (Watanabe, S.; Varsalona, F.; Yoo, Y.; Guillaume, J. P.; Bollen, A.; Shimazaki, K.; Moguilevsky, N. FEBS Letters 1998, 441, 476), which was engineered to carry mutations in correspondence of those residues, the mutants Asp225Val and Glu375Gln were expressed in CHO cells and their products purified and characterized. Unequivocal evidence about the existence of ester linkages as well as their relative contribution to the specific spectroscopic and catalytic properties of bLPO is here discussed.