In vitro characterization of a plastid terminal oxidase (PTOX).

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.     

Monoclonal antibodies against human low density lipoprotein. Stoichiometric binding studies using Fab fragments

Because of its physical properties, apolipoprotein B (apo B) has remained poorly characterized. In an attempt to elucidate apo B structure, the Fab fragments of 3 different monoclonal anti-human apo B antibodies were tested in a quantitative assay for their binding to human low density lipoprotein (LDL). In each case the assay gave a linear Scatchard plot with a maximum of 1 Fab fragment bound to a single LDL particle. This result favors an LDL model containing a single large Mr apo B protein, which is not composed of multiple, identical, small Mr subunits.     

Polyoma virus mutant with normal transforming ability but impaired tumorigenic potential.

Cloned DNA from the P155 mutant of polyoma virus transforms cells in culture as efficiently as wild-type DNA, but has a much lower tumorigenic potential when injected into newborn rats. Like cells transformed by wild-type DNA, cells transformed by the mutant DNA grow in low serum concentrations, form colonies in agar suspension, and grow to high saturation densities compared with untransformed cells. They are, however, much less tumorigenic since they transplant 100- to 2,000-fold less efficiently than cells transformed by wild-type DNA. Substitution of the region between 89.7 and 1.8 map units by the corresponding region of P155 DNA decreased the tumorigenicity of wild-type DNA. When this region was isolated from wild-type DNA and substituted in P155 DNA, the tumorigenicity of the latter increased to values comparable to those of wild-type DNA. This showed that the lesion affecting tumorigenicity occurred between 89.7 and 1.8 map units on the polyoma virus genome. Sequence analysis in this region revealed a 12-base-pair deletion between nucleotides 1,347 and 1,360. This identified P155 as an mlt mutant, i.e., a mutant with a deletion from a region which encodes parts of the large and middle T antigens.     

Androgen on the estrogen receptor I — Binding and in vivo nuclear translocation

In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C₁₉ steroids bearing a 17β and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (> 0.4mg) of 5 α aihydrotestosterone provoked $\text{in vivo}$ as $\text{in vitro}$ the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation $\text{in vivo}$ provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.     

Effect of apolipoprotein A-I lipidation on the formation and function of pre-beta and alpha-migrating LpA-I particles

A unique class of lipid-poor high-density lipoprotein, pre-beta1 HDL, has been identified and shown to have distinct functional characteristics associated with intravascular cholesterol transport. In this study we have characterized the structure/function properties of poorly lipidated HDL particles and the factors that mediate their conversion into multimolecular lipoprotein particles. Studies were undertaken with homogeneous recombinant HDL particles (LpA-I) containing apolipoprotein (apo) A-I and various amounts of palmitoyloleoylphosphatidylcholine (PC) and cholesterol. Complexation of apoA-I with small amounts of PC and cholesterol results in the formation of discrete lipoprotein structures that have a hydrated diameter of about 6 nm but contain only one molecule of apoA-I (Lp1A-I). While the molecular charge and alpha-helix content of apoA-I are unaffected by lipidation, the thermodynamic stability of the protein is reduced significantly (from 2.4 to 0.9 kcal/mol of apoA-I). Evaluation of apoA-I conformation by competitive radioimmunoassay with monoclonal antibodies shows that addition of small amounts of PC and cholesterol to apoA-I significantly increases the immunoreactivity of a number of domains over the entire molecule. Increasing the ratio of PC:apoA-I to 10:1 in the Lp1A-I complex is associated with increases in the alpha-helix content and stability of apoA-I. However, incorporation of 10-15 mol of PC destabilizes the Lp1A-I complex and promotes the formation of more thermodynamically stable (1.8 kcal/mol of apoA-I) bimolecular structures (Lp2A-I) that are approximately 8 nm in diameter. The formation of an Lp2A-I particle is associated with an increased immunoreactivity of most of the epitopes studied, with the exception of one central domain (residues 98-121), which becomes significantly less exposed. This structural change parallels a significant increase in the net negative charge on the complex. Characterization of the ability of these lipoproteins to act as substrates for lecithin:cholesterol acyltransferase (LCAT) shows that unstable Lp1A-I complexes stimulate a higher rate of cholesterol esterification by LCAT than the small but more stable Lp2A-I particles (Vmax values are 5.8 and 0.3 nmol of free cholesterol esterified/h, respectively). The ability of LCAT to interact with lipid-poor apoA-I suggests that LCAT does not need to bind to the lipid interface on an HDL particle but that LCAT may directly interact with apoA-I. The data suggests that lipid-poor HDL particles may be metabolically reactive particles because they are thermodynamically unstable.     

Travellers' malaria - 'one shoe does not fit all'

Travellers' malaria is an exciting topic. It is a field in flux with evolving options for chemoprophylaxis, self-diagnosis, self-treatment, risk/strategy analyses and surveillance. Ideologies vary and experts differ but debate is needed and can bring change. The launch of a new thematic series in the Malaria Journal -- " Travellers' malaria " -- creates an ideal forum to bring together research papers, reviews, opinion papers and commentaries, and will hopefully stimulate debate.     

Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to D-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2(-/-) mice was associated with normal levels of IL-1 beta, IL-6, and IFN-gamma, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1 beta, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2(-/-) mice displayed diminished IFN-gamma synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-gamma production required for activation of antibacterial effector mechanisms.     

High levels of cryptic species diversity uncovered in Amazonian frogs

One of the greatest challenges for biodiversity conservation is the poor understanding of species diversity. Molecular methods have dramatically improved our ability to uncover cryptic species, but the magnitude of cryptic diversity remains unknown, particularly in diverse tropical regions such as the Amazon Basin. Uncovering cryptic diversity in amphibians is particularly pressing because amphibians are going extinct globally at an alarming rate. Here, we use an integrative analysis of two independent Amazonian frog clades, Engystomops toadlets and Hypsiboas treefrogs, to test whether species richness is underestimated and, if so, by how much. We sampled intensively in six countries with a focus in Ecuador (Engystomops: 252 individuals from 36 localities; Hypsiboas: 208 individuals from 65 localities) and combined mitochondrial DNA, nuclear DNA, morphological, and bioacoustic data to detect cryptic species. We found that in both clades, species richness was severely underestimated, with more undescribed species than described species. In Engystomops, the two currently recognized species are actually five to seven species (a 150-250% increase in species richness); in Hypsiboas, two recognized species represent six to nine species (a 200-350% increase). Our results suggest that Amazonian frog biodiversity is much more severely underestimated than previously thought.