Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

The genomic ancestry of individuals from different geographical regions of Brazil is more uniform than expected

Based on pre-DNA racial/color methodology, clinical and pharmacological trials have traditionally considered the different geographical regions of Brazil as being very heterogeneous. We wished to ascertain how such diversity of regional color categories correlated with ancestry. Using a panel of 40 validated ancestry-informative insertion-deletion DNA polymorphisms we estimated individually the European, African and Amerindian ancestry components of 934 self-categorized White, Brown or Black Brazilians from the four most populous regions of the Country. We unraveled great ancestral diversity between and within the different regions. Especially, color categories in the northern part of Brazil diverged significantly in their ancestry proportions from their counterparts in the southern part of the Country, indicating that diverse regional semantics were being used in the self-classification as White, Brown or Black. To circumvent these regional subjective differences in color perception, we estimated the general ancestry proportions of each of the four regions in a form independent of color considerations. For that, we multiplied the proportions of a given ancestry in a given color category by the official census information about the proportion of that color category in the specific region, to arrive at a "total ancestry" estimate. Once such a calculation was performed, there emerged a much higher level of uniformity than previously expected. In all regions studied, the European ancestry was predominant, with proportions ranging from 60.6% in the Northeast to 77.7% in the South. We propose that the immigration of six million Europeans to Brazil in the 19th and 20th centuries--a phenomenon described and intended as the "whitening of Brazil"--is in large part responsible for dissipating previous ancestry dissimilarities that reflected region-specific population histories. These findings, of both clinical and sociological importance for Brazil, should also be relevant to other countries with ancestrally admixed populations.     

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.     

Interspecific hybridization in oysters: Restriction Enzyme Digestion Chromosome Banding confirms Crassostrea angulata × Crassostrea gigas F1 hybrids

The taxonomic status of the two commercially important cupped oysters, Crassostrea angulata, the Portuguese oyster (Lamarck, 1819) and Crassostrea gigas, the Japanese oyster (Thunberg, 1793) has long been in question. The recent observation of the hybridization between C. gigas and C. angulata and the production of fertile F1s led us to search for cytogenetic evidence of both parental genomes in the interspecific hybrids. The cytogenetic characterization of the hybrids was performed by the use of restriction endonuclease treatments. This technique has recently shown the potential for individual chromosome identification by banding in oysters. Chromosomes of C. gigas, C. angulata and their hybrids were treated with two different restriction enzymes (ApaI and HaeIII), stained with Giemsa, and examined for banding patterns. These chromosome markers allowed the parental haploid sets to be identified in the hybrids. The analysis of the banded karyotypes of the interspecific hybrids showed that for each chromosome pair, one of the homologues presented a banding pattern consistent with that of C. gigas and the other homologue presented a banding pattern consistent with that of C. angulata. These cytogenetic results substantiate the reported interspecific hybridization between C. gigas and C. angulata. In view of these results and taking into account the present expansion of C. gigas aquaculture in southern Europe, the question of the need for preservation of pure C. angulata stocks should be raised as only a few populations remain in the south of Spain and Portugal. Recently, changes in the genetic composition of populations in southern Portugal have indeed been observed, showing that human activities have created contact zones between the two taxa while no natural sympatric zones exist in Europe.     

Atheroma development in apolipoprotein E-null mice is not regulated by phosphorylation of p27(Kip1) on threonine 187

Excessive cellular proliferation is thought to contribute to neointimal lesion development during atherosclerosis and restenosis after angioplasty. Inhibition of cyclin-dependent kinase (CDK) activity by p27 inhibits mammalian cell growth. Mounting evidence indicates that p27 negatively regulates neointimal thickening in animal models of restenosis and atherosclerosis, and its expression in human neointimal lesions is consistent with such a protective role. Cell cycle progression is facilitated by cyclinE/CDK2-dependent phosphorylation of p27 on threonine 187 (T187) during late G1. The purpose of this study was to assess whether this phosphorylation event plays a role during atherosclerosis. To this end, we generated apolipoprotein E-null mice with both p27 alleles replaced by a mutated form non-phosphorylatable at T187 (apoE-/-p27T187A mice) and investigated the kinetics of atheroma development in these animals compared to apoE-/- controls with an intact p27 gene. Fat feeding resulted in comparable level of hypercholesterolemia in both groups of mice. Surprisingly, aortic p27 expression was not increased in fat-fed apoE-/-p27T187A mice compared with apoE-/- controls. Moreover, atheroma size, lesion cellularity, proliferation, and apoptotic rates were undistinguishable in both groups of fat-fed mice. Thus, in contrast to previous studies that highlight the importance of p27 phosphorylation at T187 on the control of p27 expression and function in different tissues and pathophysiological scenarios, our findings demonstrate that this phosphorylation event is not implicated in the control of aortic p27 expression and atheroma progression in hypercholesterolemic mice.     

Structural basis for the light regulation of chloroplast NADP malate dehydrogenase

The activity of chloroplast NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) in both C3 and C4 plants is regulated by light intensity. In darkness, the activity of the enzyme can be less than 1% of the maximal activity found at high light intensities. The extent of activation in the light is dynamic, responding rapidly to changes in light intensity and adapting to changes in photosynthetic rate. Enzyme activation is caused by thioredoxin-catalyzed reduction of two regulatory disulfide bonds, while inactivation is accomplished by thioredoxin-catalyzed re-oxidation. In the case of NADP-MDH, the coenzyme substrates NADP+ and NADPH modify the rate of this interconversion and seem to be important to the extent of activation in vivo. The recent determination of the X-ray structure of the oxidized, dark form of NADP-MDH from the C4 plants Flaveria bidentis and Sorghum shows how oxidation of a disulfide bond can inactivate the enzyme. This review discusses the various structural features of NADP-MDH that seem to be responsible for the regulatory properties of the enzyme and emphasizes that large changes of activity can be accomplished by multiple, small, reinforcing changes rather than a single large change in a signal molecule concentration.     

Immunoquantification and enzyme kinetics of alpha-L-iduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients.

alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A kinetic comparison between E2P and the E2P-like state induced by a beryllium fluoride complex in the Na,K-ATPase. Interactions with Rb+

Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeF_x) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeF_x is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, k_obs, for the formation of E2P decreases with [Pi] whereas that of E2BeF_x increases with [BeF_x]. This might wrongly be taken as evidence of a mechanism where the binding of BeF_x induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeF_x follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na⁺. Although E2P and E2BeF_x are able to form states with 2 occluded Rb⁺, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb⁺ is much lower in E2BeF_x than in E2P. The higher rates of Rb⁺ occlusion and deocclusion observed for E2BeF_x, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeF_x mimics the E2P ground state.