The unusual cell biology of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis

The Crenarchaeon Ignicoccus hospitalis is an anaerobic, obligate chemolithoautotrophic hyperthermophile, growing by reduction of elemental sulfur using molecular hydrogen as electron donor. Together with Nanoarchaeum equitans it forms a unique, archaeal biocoenosis, in which I. hospitalis serves as host for N. equitans. Both organisms can be cultivated in a stable coculture which is mandatory for N. equitans but not for I. hospitalis. This strong dependence is affirmed by the fact that N. equitans obtains its lipids and amino acids from the host. I. hospitalis cells exhibit several unique features: they can adhere to surfaces by extracellular appendages (‘fibers’) which are not used for motility; they use a novel CO₂ fixation pathway, the dicarboxylate/4-hydroxybutyrate pathway; and they exhibit a unique cell envelope for Archaea consisting of two membranes but lacking an S-layer. These membranes form two cell compartments, a tightly packed cytoplasm surrounded by a weakly staining intermembrane compartment (IMC) with a variable width from 20 to 1,000 nm. In this IMC, many round or elongated vesicles are found which may function as carriers of lipids or proteins out of the cytoplasm. Based on immuno-EM analyses and immuno-fluorescence experiments it was demonstrated recently that the A₁A_O ATP synthase, the H₂:sulfur oxidoreductase complex and the acetyl-CoA synthetase (ACS) of I. hospitalis are located in its outermost membrane. Therefore, this membrane is energized and is here renamed as “outer cellular membrane” (OCM). Among all prokaryotes possessing two membranes in their cell envelope, I. hospitalis is the first organism with an energized outermost membrane and ATP synthesis outside the cytoplasm. Since DNA and ribosomes are localized in the cytoplasm, energy conservation is separated from information processing and protein biosynthesis in I. hospitalis. This raises questions concerning the function and characterization of the two membranes, the two cell compartments and of a possible ATP transfer to N. equitans.     

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

Summary The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 by away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam ^– mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.     

Emergent properties and the context objection to reduction

Reductionism is a central issue in the philosophy of biology. One common objection to reduction is that molecular explanation requires reference to higher-level properties, which I refer to as the context objection. I respond to this objection by arguing that a well-articulated notion of a mechanism and what I term mechanism extension enables one to accommodate the context-dependence of biological processes within a reductive explanation. The existence of emergent features in the context could be raised as an objection to the possibility of reduction via this strategy. I argue that this objection can be overcome by showing that there is no tenable argument for the existence of emergent properties that are not susceptible to a reductive explanation.     

Species of natural hybrid origin and misinformation in the Irises: A reappraisal of the presence of I. aphylla L. in Italy

Abstract

The identity and status of the records of Iris aphylla L. for Italy are reconsidered. The authors are of the opinion that I. perrieri Simonet ex N. Service, present in Savoy, France, is genetically and morphologically distinct from I. aphylla, and that the Italian populations from Piemonte are in fact conspecific with I. perrieri. In addition, we consider that another iris, I. benacensis A. Kern. ex Stapf , which occurs near Lago di Garda (Mt. Brione) and is often also regarded as a synonym of I. aphylla, is not conspecific with either I. aphylla or I. perrieri. Macro-, micro-morphological and biosystematic data obtained during this investigation suggest a possible natural hybrid origin of these species and confirm the opinion of the authors, which is justified also by the different chromosome numbers and distribution of the taxa examined.     

Methylation of CpG dinucleotides in the lacI gene of the Big Blue® transgenic mouse

Cytosine residues at CpG dinucleotides can be methylated by endogenous methyltransferases in mammalian cells. The resulting 5-methylcytosine base may undergo spontaneous deamination to form thymine causing G/C to A/T transition mutations. Methylated CpGs also can form preferential targets for environmental mutagens and carcinogens. The Big Blue® transgenic mouse has been used to investigate tissue and organ specificity of mutations and to deduce mutational mechanisms in a mammal in vivo. The transgenic mouse contains approximately 40 concatenated lambda-like shuttle vectors, each of which contains one copy of an Escherichia coli lacI gene as a mutational target. lacI mutations in lambda transgenic mice are characterized by a high frequency of spontaneous mutations targeted to CpG dinucleotides suggesting an important contribution from methylation-mediated events. To study the methylation status of CpGs in the lacI gene, we have mapped the distribution of 5-methylcytosines along the DNA-binding domain and flanking sequences of the lacI gene of transgenic mice. We analyzed genomic DNA from various tissues including thymus, liver, testis, and DNA derived from two thymic lymphomas. The mouse genomic DNAs and methylated and unmethylated control DNAs were chemically cleaved, then the positions of 5-methylcytosines were mapped by ligation-mediated PCR which can be used to distinguish methylated from unmethylated cytosines. Our data show that most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent (>98%) in all tissues tested; only a few sites are partially (70–90%) methylated. We conclude that tissue-specific methylation is unlikely to contribute significantly to tissue-specific mutational patterns, and that the occurrence of common mutation sites at specific CpGs in the lacI gene is not related to selective methylation of only these sequences. The data confirm previous suggestions that the high frequency of CpG mutations in lacI transgenes is related to the presence of 5-methylcytosine bases.     

‘Don't be so serious’: ethical play,Islam, and the transcendent

Modes of play and playfulness are central to ethics, yet have not been as rigorously considered by anthropologists as have more earnest forms of ethical life. In this article, I argue that attention to play reframes recent anthropological debates about ethical transcendence and immanence. I do so through a consideration of the Islamic discourse of ‘calculation’ (ḥisāb), an idiom by which Muslims articulate their hoped-for state in the hereafter through the imagery of a divine accounting of good and bad deeds. Drawing on ethnography from the Indonesian province of Aceh, I show how ḥisāb cultivates forms of epistemological play through which Muslims explore the inscrutability of transcendence. Such play reveals the socially and theologically emergent qualities of transcendent truths and values, suggesting hidden affinities between transcendent stances and more immanent forms of ethical life.     

What Capabilities for the Animal?

In this essay, I defend a bi-constructivist approach to ethology—a constructivist ethology assuming that each animal adopts constructivist strategies. I put it in opposition to what I call a realist-Cartesian approach, which is currently the dominant approach to ethology and comparative psychology. The starting point of the bi-constructivist approach can be formulated as a shift from the classical Aristotelian question “What is an animal?” to the Spinozean question, which is much less classical but which seems to me to be much stronger: “What are the capacities of the animal?”. Is it possible to conceptualize an ethology which insists on interpretation and therefore on invention, innovation and creativity, rather than on causality, the monotony of behavioural routines, and/or genetic or environmental determination? Such an ethology would be based not on the fiction of an absent observer but on fully recognizing the necessity of an observer, who is effectively present in order to get an observation. A pluralistic ethology does not dissociate itself from the marginal epistemologies of practitioners like animal trainers, hunters, stockbreeders etc., or, moreover, non-western experts. An ethology of this kind is not clamped within the boundaries of purely academic epistemology, obsessed by demarcation lines between the human and the animal. My work on the bi-constructivist approach represents a contribution towards the elaboration of an authentically biosemiotic ethology, one which is significantly different from the mechanical ethology of today.     

Depth cues, behavioural context, and natural illumination: some potential limitations of video playback techniques

By expanding on issues raised by D’Eath (1998), I address in this article three aspects of vision that are difficult to reproduce in the video- and computer-generated images used in experiments, in which images of conspecifics or of predators are replayed to animals. The lack of depth cues derived from binocular stereopsis, from accommodation, and from motion parallax may be one of the reasons why animals do not respond to video displays in the same way as they do to real conspecifics or to predators. Part of the problem is the difficulty of reproducing the closed-loop nature of natural vision in video playback experiments. Every movement an animal makes has consequences for the pattern of stimulation on its retina and this ”optic flow” in turn carries information about both the animal’s own movement and about the three-dimensional structure of the environment. A further critical issue is the behavioural context that often determines what animals attend to but that may be difficult to induce or reproduce in an experimental setting. I illustrate this point by describing some visual behaviours in fiddler crabs, in which social and spatial context define which part of the visual field a crab attends to and which visual information is used to guide behaviour. I finally mention some aspects of natural illumination that may influence how animals perceive an object or a scene: shadows, specular reflections, and polarisation reflections. Received: 23 November 1999 / Received in revised form: 9 February 2000 / Accepted: 10 February 2000     

Effect of the co-existence of galactosyl and phosphomannosyl residues on β-hexosaminidase on the processing and transport of the enzyme in mucolipidosis I fibroblasts

Cultured fibroblasts from patients with the lysosomal storage disease, mucolipidosis II, produce complex glycosylated lysosomal enzymes which are preferentially excreted presumably due to the absence of specific phosphomannosyl recognition residues needed for intracellular retention. Complex glycosylated hydrolases are also produced by fibroblasts from patients with mucolipidosis I but an abnormal excretion is not apparent in this disorder. Intra- and extracellular distribution, lectin binding, and specific endocytosis were criteria used to compare the properties of intra- and extracellular β-hexosaminidase derived from mucolipidosis I and normal fibroblast cultures. Mucolipidosis I fibroblasts did not hyperexcrete β-hexosaminidase when maintained in serum-free medium. Using the specifity of ricin binding to terminal galactosyl residues, the most galactosylated forms of the enzyme derived from mucolipidosis I cell extracts and culture fluids were found in the mucolipidosis I cell extracts (50% of total enzyme). Mucolipidosis I-excreted β-hexosaminidase which was eluted from ricin-120-Sepharose, was a high-uptake form in endocytosis experiments while unbound enzyme was a low-uptake form. These data suggest that β-hexosaminidase molecules contained phosphomanosyl residues necessary for receptor-mediated endocytosis as well as galactosyl residues on the same molecule. The co-existence of complex chains with high-mannose chains did not interfere with the phosphomannose-mediated endocytosis of β-hexosaminidase nor with the retention of endogenous enzyme. We can speculate that since complex oligosaccharide chains in the mucolipidosis I cellular enzyme persist due to a sialidase deficiency, more extensive sialylation of cellular enzyme in normal fibroblasts probably occurs at some point during post-translational processing. However, the presence of sialidase in normal cells initiates complex chain trimming in the lysosomes resulting in a less glycosylated end product.     

Major histocompatibility complex of the mole-rat

The major histocompatibility complex (Mhc) is a group of loci coding for lymphocyte membrane glycoproteins that provide the context for the recognition of foreign antigens in the initial phase of the immune response. The complex contains a large number of loci, some of which are highly polymorphic. The complexity and polymorphism pose a number of questions concerning the evolution of the Mhc. In an attempt to answer some of these questions, we have begun to study the Mhc of the mole-rat, Spalax ehrenbergi, a rodent representing a complex of sibling species occupying ecologically and geographically clearly delineated regions within the borders of Israel. In an earlier publication we identified the Spalax major histocompatibility (Smh) complex serologically and biochemically. Here, we analyze the Smh by Southern blotting of DNA fragments produced by restriction enzyme digestion. The fragments were hybridized to mouse probes specific for class I, class II, and C4 genes. The analysis has revealed that the Smh complex contains as many class I genes as the mouse does and that these genes are polymorphic. The number of class II genes could not be determined with certainty, but it is probably not greater than in the mouse. Polymorphism was also detected at the loci coding for the complement component 4 (C4), which are probably closely linked to the Smh complex. The polymorphism of mole-rat class I loci contrasts with the reported monomorphism of these loci in the Syrian hamster. Since the mole-rat leads a solitary, subterranean life, as the Syrian hamster does, ecology cannot be an explanation for the lack of class I polymorphism in the latter species.On leave from the Department of Physiology, University of Zagreb Medical Faculty, Zagreb, Yugoslavia.     

A hidden beneficial: biology of the tick‐wasp Ixodiphagus hookeri in Germany

The parasitic wasp Ixodiphagus hookeri parasitizes hard ticks and is therefore considered as a potential candidate for biological control of ticks. However, there are still considerable gaps of knowledge about the biology of I. hookeri, especially for European populations. Thus, the present study was performed to assess important life‐history parameters of the parasitoid in Germany. Field studies accomplished in three successive years revealed that unfed Ixodes ricinus nymphs are infested by the parasitoid at a low but constant rate of 1.9–3.8% and that adult wasps are present only during a short period in late summer. The mean developmental time of wasps in I. ricinus nymphs ranged from 28 to 70 days under constant laboratory conditions and was prolonged in the second half of the year. Bioassays on parasitization and host preference behaviour showed that unfed nymphs of the host species I. ricinus are significantly preferred in experiments, in which unfed and engorged larvae as well as fully engorged nymphs were offered as alternatives. The marsh tick Dermacentor reticulatus was not accepted as an alternative host. Our data show that the investigated I. hookeri populations differ markedly from populations in other regions of the world in many aspects. The adaptation of different strains to local conditions explains the limited success of imported strains in earlier biological control attempts and highlights the importance of doing research to enhance the control potential of native strains.     

Mammalian DNA ligases

DNA joining enzymes play an essential role in the maintenance of genomic integrity and stability. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Since DNA ligase II appears to be derived from DNA ligase III by a proteolytic mechanism, the three LIG genes can account for the four biochemically distinct DNA ligase activities, DNA ligases I, II, III and IV, that have been purified from mammalian cell extracts. It is probable that the specific cellular roles of these enzymes are determined by the proteins with which they interact. The specific involvement of DNA ligase I in DNA replication is mediated by the non-catalytic amino-terminal domain of this enzyme. Furthermore, DNA ligase I participates in DNA base excision repair as a component of a multiprotein complex. Two forms of DNA ligase III are produced by an alternative splicing mechanism. The ubiqitously expressed DNA ligase III-α forms a complex with the DNA single-strand break repair protein XRCC1. In contrast, DNA ligase III-β, which does not interact with XRCC1, is only expressed in male meiotic germ cells, suggesting a role for this isoform in meiotic recombination. At present, there is very little information about the cellular functions of DNA ligase IV.     

Cooperative transition between two helical conformations in a linear system: Poly-L-proline I ⇌ II. I. Equilibrium studies

The solvent-induced conformational transition between the two helical forms of poly-L -proline is studied as a model for cooperative order ? order transitions. The chain length dependent equilibrium data in two solvent systems are described by Schwarz's theory, which is based upon the most general formulation of the linear Ising model with nearest neighbor interactions. The parameter σ which describes the difficulty of nucleation of a I (II) residue in an uninterrupted II (I)-helix is 10^?5 in both solvent systems. The ratios of the nucleation difficulties of states I and II at the ends of the chains β′ and β″ are very different in the two systems. Nucleation difficulty within the chain is interpreted as being due to unfavorable excess interaction energies at the I–II and II–I junctions, which add up to 7 kcal/mole of nuclei as calculated from the σ value. A similar value is computed from the atomic interactions at the junctions. In contrast to this intrinsic properly of poly-L -proline, the energies of I and II residues at the ends are heavily influenced by interactions of the endgroups with the solvent. The above values of the nucleation parameters are determined by a new least-square fitting procedure which does not necessitate the assumption of the dependence of the equilibrium constant s for propagation upon the external parameters, but yields this function from the experimental transition data. A quantitative explanation of this experimental s function through the binding of solvent is attempted. In the transition region a very small free energy change (about 0.1 kcal/mole), arising from a preferential binding of solvent molecules to one of the conformational states, is sufficient for a complete conversion from one helical form to the other.     

Identification of Photosystem I components from a glaucocystophyte,Cyanophora paradoxa: The PsaD protein has an N-terminal stretch homologous to higher plants

Thylakoid membranes and Photosystem I (PS I) complexes were isolated from a glaucocystophyte, Cyanophora paradoxa, which is thought to have the most primitive ‘plastids’, and the proteins related to PS I were examined. The intrinsic light-harvesting chlorophyll protein complexes of PS I (LHC I) were not detected by an immunological method. The PS I complexes consisted of at least eight low-molecular-mass proteins in addition to PS I reaction center proteins. The N-terminal sequence of the PsaD protein has higher homology to that of Chlamydomonas reinhardtii and land plants, than to that of other algae or cyanobacteria. On the other hand, the PsaL sequence has the highest homology to those of cyanobacteria. Taking into account the other sequences of PS I components whose genes are encoded in the cyanelle genome, and the fact that LHC I is not detected, it is concluded that PS I of C. paradoxa has chimeric characteristics of both ‘green’ lineages and cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Revising the ethnodevelopment model: addressing Karen self-determination within the context of the failed ethnocratic state of military-ruled Burma

My paper examines the Karen ethnic nationality and their fifty-eight-year self-determination struggle against ethnic cleansing resulting from the ethnocratic and military governments of Burma. I frame Karen self-determination as a development issue by employing Rodolfo Stavenhagen's ethnodevelopment model. Ethnodevelopment argues that, if asymmetrical development occurs within a multi-ethnic state, state-oriented ethnic minority development strategies are needed to neutralize the asymmetry. However, Stavenhagen's ethnodevelopment does not question the premise of an authoritarian state or the systemic crisis experienced by ethnic minorities under authoritarian rule. Thus, I revise ethnodevelopment from its top-to-bottom trajectory where ethnic minority development is dependent upon the centralized state, to a bottom-to-top trajectory I designate as liberation ethnodevelopment. I argue that Karen liberation ethnodevelopment is also a development process, but one that develops and shields the Karen from ethnic cleansing.     

Evidence of lysosomal membrane permeabilization in mucopolysaccharidosis type I: Rupture of calcium and proton homeostasis

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α‐iduronidase (IDUA), which leads to intralysosomal accumulation of glysosaminoglycans. Patients with MPS I present a wide range of clinical manifestations, but the mechanisms by which these alterations occur are still not fully understood. Genotype–phenotype correlations have not been well established for MPS I; hence, it is likely that secondary and tertiary alterations in cellular metabolism and signaling may contribute to the physiopathology of the disease. The aim of this study was to analyze Ca²⁺ and H⁺ homeostasis, lysosomal leakage of cysteine proteases, and apoptosis in a murine model of MPS I. After exposition to specific drugs, cells from Idua?/? mice were shown to release more Ca²⁺ from the lysosomes and endoplasmic reticulum than Idua+/+ control mice, suggesting a higher intraorganelle store of this ion. A lower content of H⁺ in the lysosomes and in the cytosol was found in cells from Idua?/? mice, suggesting an alteration of pH homeostasis. In addition, Idua?/? cells presented a higher activity of cysteine proteases in the cytosol and an increased rate of apoptotic cells when compared to the control group, indicating that lysosomal membrane permeabilization might occur in this model. Altogether, our results suggest that secondary alterations—as changes in Ca²⁺ and H⁺ homeostasis and lysosomal membrane permeabilization—may contribute for cellular damage and death in the physiopathology of MPS I. J. Cell. Physiol. 223: 335–342, 2010. © 2010 Wiley‐Liss, Inc.     

Site-Specific Nickase from Bacillus Species Strain D6

Three site-specific endonucleases were found in thermophilic strain Bacillus species D6. One of them, BspD6II, is an isoschizomer of Eco57I. The second, BspD6III, is present in the strain in very small amount; therefore, it has not been characterized. This paper is devoted to the third, BspD6I, which recognizes pentanucleotide site 5'-GAGTC-3' and cleaves only one DNA strand at a distance of 4 nucleotides from the site in the 3'-direction in the chain with the GAGTC sequence, i.e., it behaves as a site-specific nickase. Nickase N.BspD6I cleaves one DNA strand only in double-stranded DNA and does not cleave single-stranded DNA. Site-specific methylase SscL1I (an isohypectomer of M·HinfI) that methylates adenine in the sequence 5'-GANTC-3' prevents DNA hydrolysis by nickase BspD6I.     

Photosystem I from the unusual cyanobacterium Gloeobacter violaceus

Photosystem I (PS I) from the primitive cyanobacterium Gloeobacter violaceus has been purified and characterised. Despite the fact that the isolated complexes have the same subunit composition as complexes from other cyanobacteria, the amplitude of flash-induced absorption difference spectra indicates a much bigger antenna size with about 150 chlorophylls per P700 as opposed to the usual 90. Image analysis of the PS I preparation from Gloeobacter reveals that the PS I particles exist both in a trimeric and in a monomeric form and that their size and shape closely resembles other cyanobacterial PS I particles. However, the complexes exhibit a higher molecular weight as could be shown by gel filtration. The preparation contains novel polypeptides not related to known Photosystem I subunits. The N-terminal sequence of one of those polypeptides has been determined and reveals no homology to known or hypothetical proteins. Immunoblotting shows a cross-reaction of three of the polypeptide bands with an antibody raised against the major LHC from the diatom Cyclotella cryptica. Electron microscopy reveals a novel T-shaped complex which has never been observed in any other cyanobacterial PS I preparation. 77 K spectra of purified PS I show an extreme blue-shift of the fluorescence emission, indicating an unusual organisation of the PS I antenna system in Gloeobacter. This revised version was published online in June 2006 with corrections to the Cover Date.     

Scintillation Proximity Assay for Human DNA Topoisomerase I Using Recombinant Biotinyl-Fusion Protein Produced in Baculovirus-Infected Insect Cells

DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated duringin vivoexpression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of³H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein–DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.