Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.     

An ascaridoid nematode from a hippopotamus

A species within the genus Toxocara is reported for the first time from a hippopotamus in Uganda (Africa). Species in Toxocara are listed and differentiated from this species. The most important differentiating feature is the presence of lateral cuticular expansions in the cervical region which are more than 0·1 mm wide when viewed from the dorsal side, but extend only for one-third of the length of the oesophagus from the anterior end. Otherwise this species most closely resembles T. vitulorum, with which a detailed comparison is made.     

Estimating abundance with sparse data: tigers in northern Myanmar

As part of a national strategy for recovering tiger populations, the Myanmar Government recently proposed its first and the world’s largest tiger reserve in the Hukaung Valley, Kachin State. During November 2002–June 2004, camera-traps were used to record tigers, identify individuals, and, using capture–recapture approaches, estimate density in the reserve. Despite extensive (203 trap locations, 275–558 km² sample plots) and intensive (>4,500 trap nights, 9 months of sampling) survey efforts, only 12 independent detections of six individual tigers were made across three study sites. Due to the sparse data, estimates of tiger abundance generated by Program CAPTURE could not be made for all survey sites. Other approaches to estimating density, based on numbers of tigers caught, or derived from borrowed estimates of detection probability, offer an alternative to capture–recapture analysis. Tiger densities fall in the range of 0.2–2.2 tigers/100 km², with 7–71 tigers inside a 3,250 km² area of prime tiger habitat, where efforts to protect tigers are currently focused. Tiger numbers might be stabilized if strict measures are taken to protect tigers and their prey from seasonal hunting and to suppress illegal trade in wildlife. Efforts to monitor abundance trends in the tiger population will be expensive given the difficulty with which tiger data can be obtained and the lack of available surrogate indices of tiger density. Monitoring occupancy patterns, the subject of a separate ongoing study, may be more efficient.     

Purification and characterization of ferritin fromCampylobacter jejuni

We purified an iron-containing protein from Campylobacter jejuni using ultracentrifugation and ion-exchange chromatography. Electron microscopy of this protein revealed circular particles with a diameter of 11.5 nm and a central core with a diameter of 5.5 nm. The protein was composed of a single peptide of 21 kDa and did not serologically cross-react with horse spleen ferritin. The UV-visible spectrum of the protein showed no absorption peaks in the visible region, indicating that little or no heme is bound. The ratio of Fe:phosphate of C. jejuni ferritin was 1.5:1. From these morphological and chemical examinations, we concluded that the C. jejuni purified protein is a ferritin of the same class as that of Helicobacter pylori and Bacteroides fragilis and differs from the heme-containing bacterioferritin of Escherichia coli. The 30 N-terminal amino acids were sequenced and were found to resemble the sequences of other ferritins strongly (H. pylori ferritin, 73% identity; B. fragilis ferritin, 50% identity; E. coli gene-165 product, 50% identity), and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 26% identity; Azotobacter vinelandii, 26% identity; horse spleen ferritin 30% identity). Proteins that cross-reacted with antiserum against the ferritin of C. jejuni were found in other Campylobacter species and in H. pylori, but not in Vibrio, E. coli, or Pseudomonas aeruginosa. Received: 6 September 1994 / Accepted: 6 February 1995     

Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates

To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA.     

Complexes with the fac-{M(CO)3} (M = Tc, Re) moiety and long alkyl chain ligands as Lipiodol surrogates

Accumulation of radiopharmaceuticals in the liver is frequently observed and represents in general a limiting factor when developing novel labeled compounds for any purpose in nuclear medicine. Aiming at the treatment of liver cancer with radiopharmaceuticals, such accumulation is desired but the compounds have to remain in the liver over an extended time period rather than being washed out or redistributed over time in the whole body. Lipiodol is known to remain in the liver and we present here a study for the preparation of ¹⁸⁶Re and ^99mTc labeled Lipiodol surrogates expected to behave similarly. We have synthesized two bidentate and two tridentate ligands conjugated to a pendant C18 chain as well as their corresponding fac-[Re(CO)₃]⁺ and fac-[Tc(CO)₃]⁺ complexes. Three of the rhenium complexes have been structurally characterized. Labelling with [¹⁸⁶Re(OH₂)₃(CO)₃]⁺ and [^99mTc(OH₂)₃(CO)₃]⁺, respectively, gave yields in the range of 90%. The complexes could be extracted into Lipiodol due to their high lipophilicity and close structural relationship with the major components of Lipiodol. The complexes are stable in water and in Lipiodol for more than 24 h. These Lipiodol surrogates present new low-valent technetium and rhenium complexes for applications in liver cancer imaging and therapy.     

Health and Human Rights in Eastern Myanmar after the Political Transition: A Population-Based Assessment Using Multistaged Household Cluster Sampling

BackgroundMyanmar transitioned to a nominally civilian parliamentary government in March 2011. Qualitative reports suggest that exposure to violence and displacement has declined while international assistance for health services has increased. An assessment of the impact of these changes on the health and human rights situation has not been published.ConclusionThis large survey of health and human rights demonstrates that two years after political transition, vulnerable populations of eastern Myanmar are less likely to experience human rights violations compared to previous surveys. However, access to health services remains constrained, and risk of disease and death remains higher than the country as a whole. Efforts to address these poor health indicators should prioritize support for populations that remain outside the scope of most formal government and donor programs.     

Assessing the status of three mangrove species restored by the local community in the cyclone-affected area of the Ayeyarwady Delta,Myanmar

This paper assesses the extent of success and failure of mangrove plantations in Myanmar, restored by local people with the help of foresters under a community forestry program initiated in 1995. The species of these restored plantations are Avicennia officinalis, Avicennia marina and Heritiera fomes, each of which was restored on two plots, one on low and one on high ground, yielding a total of six plots. These plots have been continuously monitored in order to investigate survival and growth rates. The plots were established on abandoned land that had been previously used for paddy cultivation. Cyclone Nargis hit these plantations during the monitoring period, at the beginning of May, 2008. As a consequence, the survival rates of A. officinalis on low ground and A. marina on high ground declined slightly, but the overall affect was not severe. Excluding individuals affected by the cyclone, height and diameter growth of A. officinalis and A. marina were significantly higher on low ground than on high ground, i.e. on sites thought to be consistently similar to the natural habitats of these species. Contrary to these two Avicennia species, the height growth of H. fomes was higher on high ground than on low ground; the diameter growth was not significantly different. As the growth of H. fomes was very slow, however, it is still not possible to describe the differences clearly. This study may provide useful guidelines for foresters and local people to establish successful mangrove restorations and to predict production from community-owned mangrove forests.     

Monitoring surface chemical changes in the bacterial cell wall: multivariate analysis of cryo-x-ray photoelectron spectroscopy data

Gram-negative bacteria can alter the composition of the lipopolysaccharide (LPS) layer of the outer membrane as a response to different growth conditions and external stimuli. These alterations can, for example, promote attachment to surfaces and biofilm formation. The changes occur in the outermost layer of the cell and may consequently influence interactions between bacterial cells and surrounding host tissue, as well as other surfaces. Microscopic analyses, fractionation of bacterial cells, or other traditional microbiological assays have previously been used to study these alterations. These methods can, however, be time consuming and do not always give detailed chemical information about the bacterial cell surface. We here present an analytical method that provides chemical information on the outermost portion of bacterial cells with respect to protein, peptidoglycan, lipid, and polysaccharide content. The method involves cryo-x-ray photoelectron spectroscopy analyses of the outermost portion (within ～10 nm of the surface) of intact bacterial cells followed by a multivariate curve resolution analysis of carbon spectra. It can be used as a tool for characterizing and monitoring variations in the chemical composition of bacterial cell walls or of isolated outer membrane vesicles, variations that result from e.g. mutations or external stimuli. The method enabled us to predict accurately the alterations in polysaccharide content and surface chemistries of a set of well characterized Escherichia coli LPS mutants. The described approach may moreover be applied to monitor surface chemical composition of other biological samples.