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Repetitive excessive alcohol intoxication leads to neuronal damage and brain shrinkage. We examined cytoskeletal protein expression in human post-mortem tissue from Brodmann&rsquo, s area 9 of the prefrontal cortex (PFC). Brain samples from 44 individuals were divided into equal groups of 11 control, 11 alcoholic, 11 non-alcoholic suicides, and 11 suicide alcoholics matched for age, sex, and post-mortem delay. Tissue from alcoholic cohorts displayed significantly reduced expression of &alpha, and &beta, tubulins, and increased levels of acetylated &alpha, tubulin. Protein levels of histone deacetylase-6 (HDAC6), and the microtubule-associated proteins MAP-2 and MAP-tau were reduced in alcoholic cohorts, although for MAPs this was not significant. Tubulin gene expressions increased in alcoholic cohorts but not significantly. Brains from rats administered alcohol for 4 weeks also displayed significantly reduced tubulin protein levels and increased &alpha, tubulin acetylation. PFC tissue from control subjects had reduced tubulin protein expression that was most notable from the sixth to the eighth decade of life. Collectively, loss of neuronal tubulin proteins are a hallmark of both chronic alcohol consumption and natural brain ageing. The reduction of cytosolic tubulin proteins could contribute to the brain volumetric losses reported for alcoholic patients and the elderly.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and 3H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼75–80 kDa, ∼95–100 kDa, and ∼155–160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

The bacterial protein RelE severely restricts prokaryotic cell growth, probably by acting as a global inhibitor of translation. It is ubiquitous in prokaryotes as part of the RelE–RelB toxin–antitoxin system, and may be activated by nutritional stress. When the relE gene from Escherichia coli was expressed inducibly in a human osteosarcoma cell line, it was shown to retard growth and to lead to cell death by apoptosis. RelE is therefore unusual among bacterial toxins in possessing broad activity against both prokaryotes and eukaryotes, perhaps by acting on evolutionarily conserved components of the translation machinery.

We consider the hp-version of the discontinuous Galerkin (DG) time-stepping method for linear parabolic problems with non-symmetric elliptic spatial operators. We derive new analyticity estimates for the exact solutions by means of semigroup techniques. These estimates allow us to show that the hp-DG time-stepping method can resolve start-up singularities at exponential rates of convergence.

A fully discrete hp-finite element method (FEM) is presented. It combines the features of the standard hp-FEM (conforming Galerkin formulation, variable order quadrature schemes, geometric meshes, static condensation) and of the spectral element method (special shape functions and spectral quadrature techniques). The speed-up (relative to standard hp elements) is analyzed in detail both theoretically and computationally.

The Streamline Diffusion Finite Element Method (SDFEM) for a two-dimensional convection–diffusion problem is analyzed in the context of the hp-version of the Finite Element Method (FEM). It is proved that the appropriate choice of the SDFEM parameters leads to stable methods on the class of "boundary layer meshes", which may contain anisotropic needle elements of arbitrarily high aspect ratio. Consistency results show that the use of such meshes can resolve layer components present in the solutions at robust exponential rates of convergence. We confirm these theoretical results in a series of numerical examples.

Two hp–finite element methods for the Stokes problem in polygonal domains are presented: We discuss the S k × S k−2 elements which are stable on anisotropic and irregular meshes and introduce a stabilized Galerkin Least Squares approach featuring equal-order interpolation in the velocity and the pressure. Both methods lead to exponential rates of convergence provided that the data is piecewise analytic. Numerical studies on an L-shaped domain confirm these theoretical results.

Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5' end. Here, experimental evidence is presented for the existence of metastable structures in the 5' leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5' ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription.

The stable Galerkin formulation and a stabilized Galerkin least squares formulation for the Stokes problem are analyzed in the context of the hp -version of the finite element method. Theoretical results for both formulations establish exponential rates of convergence under realistic assumptions on the input data. We confirm these results by a series of numerical experiments on an L -shaped domain where the solution exhibits corner singularities.

We study convergence of the FEM for a 3D-problem of rigid scattering. Using separation of variables, analytical results are presented for stability and error estimation for an h-version Galerkin FEM with mesh-refinement in radial direction. We prove an upper bound that contains a pollution term similar to previous results in 1D and 2D. This estimate is evaluated computationally using a 3D FEM-IEM (infinite elements) implementation. The tabulated and plotted errors show the theoretically predicted pollution effect in the FE-error.

A general 2D-hp-adaptive Finite Element (FE) implementation in Fortran 90 is described. The implementation is based on an abstract data structure, which allows to incorporate the full hp-adaptivity of triangular and quadrilateral finite elements. The h-refinement strategies are based on h2-refinement of quadrilaterals and h4-refinement of triangles. For p-refinement we allow the approximation order to vary within any element. The mesh refinement algorithms are restricted to 1-irregular meshes. Anisotropic and geometric refinement of quadrilateral meshes is made possible by additionally allowing double constrained nodes in rectangles. The capabilities of this hp-adaptive FE package are demonstrated on various test problems.

In this paper we analyze the performance of the hp-Finite Element Method for a cylindrical shell problem. Our theoretical investigations show that the hp approximation converges exponentially, provided that boundary layers stemming from the edge effect are resolved. The numerical results illustrate the mesh independence of the exponential convergence of the hp-FEM.

This work is devoted to a study and summary of different Infinite Element (IE) formulations for Helmholtz problems in arbitrary exterior domains. The theoretical setting for each of the different formulations is presented and related to the mathematical existence theory. The influence of a bilinear or a sesquilinear formulation is discussed as well as possible extensions to other elements. The implementation of the Infinite Element Method (IEM) incorporates the use of 2D and 3D hp Finite Elements and allows for hp-adaptive refinements. Numerical results show the computational efficiency of the coupled Finite-Infinite Element methodology.

This work is devoted to a convergence and performance study of finite-infinite element discretizations for the Helmholtz equation in exterior domains of arbitrary shape. The proposed approximation applies to arbitrary geometries, combining an hp-FE discretization between the object and a surrounding sphere and an hp infinite element (IE) discretization outside the sphere with a spectral-like representation (resulting from the separation of variables) in the “radial” direction. The described approximation is an extension of our earlier work, which was restricted to domains with separable geometry. The numerical experiments are confined to these geometrical configurations: a sphere, a (finite) cylinder, and a cylinder with spherical incaps, all within a truncating sphere. The sphere problem admits an exact solution and serves as a basis for the convergence study. Solutions to the other two problems are compared with those obtained using the boundary element method.

To the best knowledge of the authors, this work presents the first convergence analysis for the Infinite Element Method (IEM) for the Helmholtz equation in exterior domains. The approximation applies to separable geometries only, combining an arbitrary Finite Element (FE) discretization on the boundary of the domain with a spectral-like approximation in the “radial” direction, with shape functions resulting from the separation of variables. The principal idea of the presented analysis is based on the spectral decomposition of the problem.

The Helmholtz equation in a three-dimensional plate is approximated by a hierarchy of two-dimensional models. Computable a posteriori error estimators of the modeling error in exponentially weighted norms are derived, and sharp, computable estimates for their effectivity indices are also obtained. The necessity of including, besides polynomials, a certain number of trigonometric director functions into the Ansatz, in order to prevent pollution effects at high wave numbers is demonstrated both theoretically and computationally.

This work is devoted to a convergence study of infinite element (IE) discretizations for the Helmholtz equation in exterior domains. The different behavior of the conjugated and the unconjugated IE formulation is analyzed in context of 1. (1) a formulation following a mathematical existence theory by Leis, 2. (2) a formulation not based on an available existence theory following Burnett. Four variational formulations are presented and the differences in implementing them are discussed. The effect of using or not using the complex conjugate in the weak formulation is carefully studied. The numerical and theoretical results clearly indicate which of the four presented formulations is the most efficient that can still give reliable results.

A stabilized mixed hp -Finite Element Method (FEM) of Galerkin Least Squares type for the Stokes problem in polygonal domains is presented and analyzed. It is proved that for equal order velocity and pressure spaces this method leads to exponential rates of convergence provided that the data is piecewise analytic.

This work is devoted to a convergence study for infinite element discretizations for Laplace and Helmholtz equations in exterior domains. The proposed approximation applies to separable geometries only, combining an hp FE discretization on the boundary of the domain with a spectral-like representation (resulting from the separation of variables) in the ‘radial’ direction. The presentation includes a convergence proof for the Laplace equation and a stability analysis for the variational formulation of the Helmholtz equation in weighted Sobolev spaces. The theoretical investigations are verified and illustrated with numerical examples for the exterior spherical domain.

It has been shown that premature translation of the plasmid-mediated toxin in hok/sok of plasmid R1 and pnd/pndB of plasmid R483 is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins at the 5'-end of the mRNA. Here, an experimental approach is presented, which allows the accurate measurement of the refolding kinetics of the 5'-end RNA fragments in vitro without chemically modifying the RNA. The method is based on acid denaturation followed by a pH-jump to neutral pH as a novel way to trap kinetically favoured RNA secondary structures, allowing the measurement of a wide range of biologically relevant refolding rates, with or without the use of standard stopped-flow equipment. The refolding rates from the metastable to the stable conformation in both the hok74 and pnd58 5'-end RNA fragments were determined by using UV absorbance changes corresponding to the structural rearrangements. The measured energy barriers showed that the refolding path does not need complete unfolding of the metastable structures before the formation of the final structures. Two alternative models of such a pathway are discussed.

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame of 33 codons designated fst. The results presented here demonstrate that the peptide encoded by fst is the par toxin. The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the fst-encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem-loop structure at the 5' end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fold increase in production of the fst-encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of fst in plasmid-free cells.

Toxin-antitoxin systems encoded by bacterial plasmids and chromosomes specify two proteins, a cytotoxin and an antitoxin. The antitoxins neutralize the cognate toxins by forming tight complexes with them. The antitoxins are unstable due to degradation by cellular proteases (Lon or Clp), whereas the toxins are stable. Here we show that orf7 (denoted relBP307) and orf6 (denoted relEP307) of Escherichia coli plasmid P307 are homologous to the relBE genes of E. coli and constitute a two-component toxin-antitoxin system: (i) relEP307 encodes a cytotoxin lethal or inhibitory to host cells; (ii) relBP307 encodes an antitoxin that prevents the lethal action of the relE-encoded toxin; (iii) RelBP307 antitoxin is degraded by Lon protease; (iv) RelBP307 antitoxin autoregulates the relBE operon of P307 at the level of transcription; (v) RelEP307 toxin acts as a co-repressor of transcription; and (vi) the relBE system stabilizes a mini-P307 replicon by the killing of plasmid-free cells. Using database searching, we found relBE homologues on the chromosomes of many Gram-negative and Gram-positive bacteria. Even more surprising, numerous relBE-homologous gene systems are present on the chromosomes of Archae. Thus, toxin-antitoxin systems homologous with relBE of E. coli are ubiquitous in prokaryotic organisms.

The present work is motivated by the new concept on various infinite elements, [Burnett, 1994], [Astley, Macaulay and Coyette, 1994], [Cremers, Fyfe and Coyette, 1994], [Cremers and Fyfe, 1995], [Gerdes and Demkowicz, 1996] and [Gerdes, 1996]. These new IEM are extensions of the original IEM, which was introduced by Bettess [Bettess, 1992].

The pediatric organ donor presents some special management problems not generally seen in the adult organ/tissue donor. The psychological and emotional impact of working with a pediatric donor many times differs significantly from the adult donor scenario, but can be a rewarding experience for the nurse and organ procurement specialist (OPS).

The hok/sok locus of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNAs, Hok mRNA and Sok antisense RNA. Hok mRNA encodes the Hok killer protein of 52 amino acid residues. Expression of hok is regulated post-transcriptionally by Sok antisense RNA. Killing of plasmid-free daughter-cells by the hok/sok system is accomplished through differential decay of the Hok and Sok-RNAs: Hok mRNA is very stable while Sok-RNA decays rapidly, thus leading to derepression of Hok mRNA translation in plasmid-free segregants, ensuring a rapid and selective killing of these cells. Sok antisense RNA is complementary to the leader region of the Hok mRNA. However, the region of complementarity does not overlap with the hok Shine-Dalgarno sequence. Thus, Sok-RNA regulates hok translation indirectly by an as yet unknown mechanism. We show here that Sok antisense RNA regulates the translation of another reading frame located in the hok/sok locus. This new reading frame, which overlaps with almost the entire hok gene, was denoted mok (mediation of killing). Point-mutations that prevent mok translation through the hok translational initiation region abolish efficient expression of hok. Furthermore, these mutations abolish the Sok-RNA-mediated control of hok gene expression. Hence, the antisense-RNA-mediated regulation of the hok gene seems to occur via translational coupling between the hok and mok reading-frames.

This paper discusses the development and content of a package of late Jurassic-early Cretaceous synrift sediments from the Bjornoya Basin. The discussion is based on the result of a detailed structural and seismostratigraphic interpretation of seismic data, limited well data from the area and models for synrift sedimentation developed from extensive field studies in well-exposed rift zones. Structural maps were constructed at two scales to elucidate the first order (regional) and second order (local) structural controls on deposition. The approximate water depths and inclination of depositional surfaces were estimated using backstripped sections tied to well data. These techniques produced an estimate of the basin architecture during deposition of the synrift megasequence. The synrift megasequence was then subdivided into five seismic sequences. This subdivision depended largely on a high resolution seismic survey of limited areal extent. Each sequence was then subdivided into seismic facies units and isochron and facies maps were constructed for each sequence. A correlation between seismic facies and lithology was made using the only available well in the area. The resultant maps were then interpreted using information on the controls on synrift sedimentation gleaned from extensive fieldwork in East Africa, Greece and the Gulf of Suez and well-constrained subsurface examples. Depositional models for each sequence were generated and areas most likely to collect reservoir prone sediments were highlighted. The main critical factors concerning the predictive nature of the study are the quality of the tie between lithology and seismic character and the assumption of a coarse grained sediment supply.

The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin. Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing. We show here that the srnB and pnd systems mediate efficient stabilization of a mini-R1 test-plasmid. We also show that the killer genes srnB' and pndA are regulated by antisense RNAs, and that the srnC- and pndB-encoded antisense RNAs, denoted SrnC- and PndB-RNAs, are unstable molecules of approximately 60 nucleotides. The srnB and pndA mRNAs were found to be very stable. The differential decay rates of the inhibitory antisense RNAs and the killer-gene-encoding mRNAs explain the induction of these gene systems by rifampicin. Furthermore, the observed plasmid-stabilization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid-free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated into killer proteins, thus leading to selective killing of the plasmid-free segregants. Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hok/sok system, despite a 50% dissimilarity at the level of DNA sequence.

The seven members of the hok killer gene family in Gram-negative bacteria are described here. The members of this gene family have been sequenced and include hok/sok from plasmid R1, flm and srnB from plasmid F, pnd from plasmids R483 and R16, and gef and relF, which are located on the Escherichia coli chromosome. The killer proteins encoded by these loci are highly toxic polypeptides of 50 to 52 amino acids. The proteins kill the cells from the inside by interfering with a vital function in the cell membrane. On the basis of their relatedness, the killer proteins and their corresponding loci are divided into four subfamilies. The members of one subfamily, hok/sok and flm, mediate plasmid maintenance by killing plasmid-free cells. The pnd and srnB subfamilies were discovered through their abilities to cause membrane damage and degradation of stable RNA. gef and relF, which constitute the chromosomal subfamily, were found because of their sequence similarity at the DNA and protein levels with other members of the hok gene family. However, no function has been described for the proteins belonging to this subfamily. Although the four subfamilies are distantly related in terms of DNA and protein sequence similarity, the overall genetic organization of the different loci has been well conserved during evolution. The expression of all of the members of the hok gene family is regulated post-transcriptionally. Thus, the expression of the hok and flm genes is regulated by small antisense RNAs that inhibit the translation of the stable hok and flm mRNAs. On the basis of structural and functional similarities, we suggest that each of the related plasmid-encoded killer genes is regulated by antisense RNAs. The conservation of this widespread gene family in Gram-negative bacteria suggests that the genes are important to the genomes that carry them.

The Helmholtz equation in a three-dimensional plate is approximated by a hierarchy of two-dimensional models. The necessity of including, besides polynomials, a certain number of trigonometric director functions into the Ansatz, in order to prevent pollution effects at high wave numbers is demonstrated for exponentially weighted norms.

Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time of cells without plasmids. The pBR322 derivative carries a fusion between part of the atp operon of Escherichia coli and the bacteriophage lambda pR promoter, and the cI857 repressor gene. The insertion of sop+ from the F plasmid or parB+ from the R1 plasmid reduced the loss frequency by a factor of 10(3) for the pBR322 derivative and by at least a factor of 10(2) for the mini-R1 plasmid. Insertion of parA+ from the R1 plasmid decreased the loss frequency of the pBR322 derivative by a factor of 10 and that of the mini-R1 plasmid by a factor of 50. When ccd+ from the F plasmid was inserted, the loss frequency of the pBR322 derivative was decreased by a factor of 10, but it had only a marginal effect on the stability of the mini-R1 plasmid. In no case was any significant structural instability of the plasmids observed.

The stability locus, parA+, of plasmid R1 is shown to be localized within a 1500 base-pair region of DNA on the largest EcoRI restriction fragment of plasmid R1. The nucleotide sequence of the region revealed the presence of two open reading frames, one of 320 codons, and another of 60 codons. The larger open reading frame encodes a polypeptide of 36,000 Mr. Deletions covering the promoter distal end of the 36,000 Mr reading frame give rise to synthesis of large amounts of truncated protein. Construction of promoter fusions between the parA+ promoter and the lacZ gene showed that the parA+ region encodes a factor that negatively regulates the expression of the 36,000 Mr protein. The locus exerting parA+-associated incompatibility, denoted incA+, was mapped to a 60 base-pair region covering the parA+ promoter. Most likely, this region is involved both in the negative regulation of the parA+ operon and in the parA+-associated incompatibility. Two explanations are suggested to explain this possible dual function of the parA+ promoter region. The parA+ region was cloned into an unstably inherited (par-) derivative of a mini-F derivative. The low copy number plasmid mini-F devoid of its own partition genes was stabilized more than 100-fold by carrying the parA+ genes. This observation is in accordance with the proposal that the parA+ locus specifies the true partition function of plasmid R1.