Spectral, electrochemical, and catalytic properties of a homologous series of manganese porphyrins as cytochrome P450 model: the effect of the degree of beta-bromination

A homologous series of β-brominated porphyrins derived from meso-tetrakis(4-carbomethoxyphenyl)porphyrinatomanganese(III) chloride, i.e., Mn(III)(Br_xTCMPP)Cl (x = 0, 2, 4, 6, and 8), was prepared and investigated as cytochrome P450 models. Hydroxylations of cyclohexane by iodosylbenzene (PhIO) and iodobenzene diacetate (PhI(OAc)₂) in the presence or absence of water were carried out as P450 model reactions. The influence of the degree of β-bromination of the macrocycle on the UV-vis spectra, the Mn(III)/Mn(II) reduction potential, and the catalytic properties of the Mn(III)(Br_xTCMPP)Cl (x = 0, 2, 4, 6, and 8) series were examined. The catalytic efficiency does not correlate with the Mn(III)/Mn(II) reduction potential and shows a bell-shaped behavior, where the best results are achieved with the hexabrominated complex. Better hydroxylation yields were achieved by using PhI(OAc)₂ as oxygen donor, but at expenses of catalyst recovery; addition of water to this system resulted in a increase in the reaction rate. Recycling of the more oxidatively robust complexes Mn(III)(Br₆TCMPP)Cl and Mn(III)(Br₈TCMPP)Cl is feasible when using PhIO as oxygen donor. Selectivity and UV-vis data suggested that hydroxylation by both PhIO and PhI(OAc)₂ share closely related active species and mechanism. We also show that the Mn(III)/Mn(II) reduction potentials are inappropriate predictors of P450-type activity of Mn porphyrin-catalyzed oxidations.     

Discrimination of conspecific faecal chemicals and spatial decisions in juvenile Iberian rock lizards (<Emphasis Type="Italic">Lacerta monticola</Emphasis>)

Faeces play a role in intraspecific chemical communication in many vertebrates, including lizards. Here, we hypothesised that juvenile Iberian rock lizards (Lacerta (=Iberolacerta) monticola) use substrate-borne conspecific faecal chemicals to assess the social environment and base their spatial decisions. To test this hypothesis, we prepared chemical stimuli by dissolving faeces in an organic solvent (dichloromethane, DCM) and conducted chemosensory trials where we quantified latency to the first tongue flick (TF) and TF rates when young captive-borne juveniles were placed in an arena compartment whose substrate was labelled by (1) a control (DCM), (2) their own faecal chemicals, (3) faecal chemicals of another juvenile, (4) faecal chemicals of an adult female or (5) faecal chemicals of an adult male. Following TF observations, we removed a partition that separated the labelled compartment from a chemically unlabelled one, and recorded for 5 min when juveniles first crossed to the unlabelled compartment and total time spent in the labelled compartment. Each juvenile was tested with all stimuli in a randomised order. In addition, juveniles belonged to different families and were unrelated to and unfamiliar with faeces donors. Taken together, TF and spatial responses toward stimuli indicate that juveniles discriminated between faecal chemicals of conspecific juveniles, adult females and males, and that they avoided remaining in substrates labelled by adult male faecal chemicals. We suggest that juveniles assess the social environment based on conspecific faecal chemicals thus avoiding aggression and cannibalistic risks undertaken from encounters with adult males.     

The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis

Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.     

Novel lipoarabinomannan-like lipoglycan (CdiLAM) contributes to the adherence of <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> to epithelial cells

The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes. Members of this group have a characteristic cell envelope structure composed primarily of branched long-chain lipids, termed mycolic acids, and a rich number of lipoglycans such as lipoarabinomanans (LAM) and lipomannans. In this study, we identified a novel LAM variant isolated from Corynebacterium diphtheriae named CdiLAM. The key structural features of CdiLAM are a linear α-1→6-mannan with side chains containing 2-linked α-D-Manp and 4-linked α-D-Araf residues. The polysaccharide backbone is linked to a phosphatidylinositol anchor. In contrast to the LAMs of other members of actinomycetales, CdiLAM presents an unusual substitution at position 4 of α-1→6-mannan backbone by α-D-Araf. Unlike the non-fimbrial adhesin 62–72p, CdiLAM did not function as a hemagglutinin to human red blood cells. Experimental evidences pointed to CdiLAM as an adhesin of C. diphtheriae to human respiratory epithelial cells, thereby, contributing to the pathogenesis of diphtheria.     

Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.     

Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity.

Programmed cell death by apoptosis of unnecessary or potentially harmful cells is clearly beneficial to multicellular organisms. Proper functioning of such a program demands that the removal of dying cells proceed without an inflammatory reaction. Phosphatidylserine (PS) is one of the ligands displayed by apoptotic cells that participates in their noninflammatory removal when recognized by neighboring phagocytes. PS ligation induces the release of transforming growth factor-beta (TGF-beta), an antiinflammatory cytokine that mediates the suppression of macrophage-mediated inflammation. In Hydra vulgaris, an organism that stands at the base of metazoan evolution, the selective advantage provided by apoptosis lies in the fact that Hydra can survive recycling apoptotic cells by phagocytosis. In unicellular organisms, it has been proposed that altruistic death benefits clonal populations of yeasts and trypanosomatids. Now we show that advantageous features of the apoptotic process can operate without death as the necessary outcome. Leishmania spp are able to evade the killing activity of phagocytes and establish themselves as obligate intracellular parasites. Amastigotes, responsible for disease propagation, similar to apoptotic cells, inhibit macrophage activity by exposing PS. Exposed PS participates in amastigote internalization. Recognition of this moiety by macrophages induces TGF-beta secretion and IL-10 synthesis, inhibits NO production, and increases susceptibility to intracellular leishmanial growth.     

Homozygosity mapping of Portuguese and Japanese forms of ataxia-oculomotor apraxia to 9p13, and evidence for genetic heterogeneity

Ataxia with oculomotor apraxia (AOA) is characterized by early-onset cerebellar ataxia, ocular apraxia, early areflexia, late peripheral neuropathy, slow progression, severe motor handicap, and absence of both telangiectasias and immunodeficiency. We studied 13 Portuguese families with AOA and found that the two largest families show linkage to 9p, with LOD scores of 4.13 and 3.82, respectively, at a recombination fraction of 0. These and three smaller families, all from northern Portugal, showed homozygosity and haplotype sharing over a 2-cM region on 9p13, demonstrating the existence of both a founding event and linkage to this locus, AOA1, in the five families. Three other families were excluded from this locus, demonstrating nonallelic heterogeneity in AOA. Early-onset cerebellar ataxia with hypoalbuminemia (EOCA-HA), so far described only in Japan, is characterized by marked cerebellar atrophy, peripheral neuropathy, mental retardation, and, occasionally, oculomotor apraxia. Two unrelated Japanese families with EOCA-HA were analyzed and appeared to show linkage to the AOA1 locus. Subsequently, hypoalbuminemia was found in all five Portuguese patients with AOA1 with a long disease duration, suggesting that AOA1 and EOCA-HA correspond to the same entity that accounts for a significant proportion of all recessive ataxias. The narrow localization of AOA1 should prompt the identification of the defective gene.     

Phytoplankton diversity and cyanobacterial dominance in a hypereutrophic shallow lake with biologically produced alkaline pH

In this work, we have characterized the diversity of phytoplanktonic species in a highly alkaline and hypereutrophic shallow lake, Santa Olalla (southwestern Spain), the evolution of their relative abundances, and that of several physicochemical parameters over 2 years. In the absence of an external input of alkaline water, Santa Olallas stable high pH (average pH 9.52, with several maxima >10.5) is explained by an extremely high photosynthetic primary productivity. A variety of phytoplankton species was observed even during pH maxima. These included several species of green algae, diatoms, and euglenoids and several cyanobacteria from the orders Nostocales and Chroococcales. Quantitatively, cyanobacteria dominated. A blooming event due to Aphanothece clathrata was observed at one pH maximum, during which the diversity as measured by the Shannon-Weaver index was extremely low. Santa Olallas cyanobacteria are alkaliphilic and/or extremely alkalitolerant and appear to be responsible for the generation and maintenance of stable high-pH conditions in their environment.Communicated by W.D. Grant