Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aß ligation to ß1-integrin conformers.

Correction to: Cell Death and Differentiation (2015) 22, 921­934; doi:10.1038/cdd.2015.5; published online 20 February 2015. Since the publication of this paper, the authors have noticed the y-axis label of Figure 7e was incorrect. It should be % of the fESP slope. This has now been rectified and the corrected article appears in this issue together with this corrigendum.

Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aß ligation to ß1-integrin conformers.

The accumulation of amyloid-ß protein (Aß) is an early event associated with synaptic and mitochondrial damage in Alzheimer's disease (AD). Recent studies have implicated the filamentous actin (F-actin) severing protein, Cofilin, in synaptic remodeling, mitochondrial dysfunction, and AD pathogenesis. However, whether Cofilin is an essential component of the AD pathogenic process and how Aß impinges its signals to Cofilin from the neuronal surface are unknown. In this study, we found that Aß42 oligomers (Aß42O, amyloid-ß protein 1-42 oligomers) bind with high affinity to low or intermediate activation conformers of ß1-integrin, resulting in the loss of surface ß1-integrin and activation of Cofilin via Slingshot homology-1 (SSH1) activation. Specifically, conditional loss of ß1-integrin prevented Aß42O-induced Cofilin activation, and allosteric modulation or activation of ß1-integrin significantly reduced Aß42O binding to neurons while blocking Aß42O-induced reactive oxygen species (ROS) production, mitochondrial dysfunction, depletion of F-actin/focal Vinculin, and apoptosis. Cofilin, in turn, was required for Aß42O-induced loss of cell surface ß1-integrin, disruption of F-actin/focal Talin-Vinculin, and depletion of F-actin-associated postsynaptic proteins. SSH1 reduction, which mitigated Cofilin activation, prevented Aß42O-induced mitochondrial Cofilin translocation and apoptosis, while AD brain mitochondria contained significantly increased activated/oxidized Cofilin. In mechanistic support in vivo, AD mouse model (APP (amyloid precursor protein)/PS1) brains contained increased SSH1/Cofilin and decreased SSH1/14-3-3 complexes, indicative of SSH1-Cofilin activation via release of SSH1 from 14-3-3. Finally, genetic reduction in Cofilin rescued APP/Aß-induced synaptic protein loss and gliosis in vivo as well as deficits in long-term potentiation (LTP) and contextual memory in APP/PS1 mice. These novel findings therefore implicate the essential involvement of the ß1-integrin-SSH1-Cofilin pathway in mitochondrial and synaptic dysfunction in AD.

Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains.

A simple and versatile system has been developed using a new cloning vector which can serve as a vehicle for integrating DNA fragments, which direct the expression of heterologous antigens, into the aroC gene on the Salmonella chromosome. The system is based on Escherichia coli plasmid vectors which contain the DNA fragment, cloned from the chromosome of S. typhimurium C5, which encodes the aroC gene. The aroC gene was modified using synthetic oligodeoxyribonucleotides so that it contained several unique restriction sites into which DNA, directing the expression of heterologous antigens, could be cloned. DNA was integrated into the S. typhimurium chromosome at aroC by transferring the vectors into S. typhimurium polA mutants and allowing homologous recombination to occur between the cloned and chromosomal aroC genes. The vectors were used to integrate nucleotide sequences into the S. typhimurium chromosome which directed the expression of tetanus toxin fragment C and the Treponema pallidum lipoprotein. The expression of both antigens was detected by Western blotting.

A mgl-like operon in Treponema pallidum, the syphilis spirochete.

A 38-kDa lipoprotein of Treponema pallidum subsp. pallidum (T. pallidum), the syphilis spirochete, previously was identified as a putative homolog of E. coli MglB [Becker et al. (1994) Infect. Immun. 62, 1381-1391]. In the present study, genome walking in regions adjacent to the T. pallidum 38-kDa lipoprotein gene has identified three contiguous genes (tp-mglB [formerly tpp38], tp-mglA, and tp-mglC) which appear to comprise a mgl-like operon in T. pallidum. A prominent transcript corresponding to tp-mglB, the first gene of the operon which encodes the carbohydrate receptor, is synthesized by T. pallidum along with lesser abundant transcript(s) corresponding to the entire T. pallidum mgl operon. An active promoter 135 bp upstream of tp-mglB is believed to direct mRNA synthesis for the operon. This is the first membrane protein-encoding operon of T. pallidum for which a putative function (glucose import) has been assigned. Furthermore, by analogy with E. coli MglB which interacts with the sensory transducer Trg to induce a chemotactic response, it is possible that T. pallidum also contains a homolog of E. coli Trg or other methyl-accepting chemotaxis proteins. The existence of a mgl operon in T. pallidum thus may have important implications with respect to T. pallidum survival, tissue dissemination, and sensory transduction during virulence expression.

The use of specific antiserum induced by lectin-antigen complexes to investigate the outer membrane antigens of Neisseria gonorrhoeae.

Extracts of gonococcal surface antigens, examined by crossed affinity electrophoresis (CAE) with wheat germ agglutinin (WGA), yielded a single antigen-lectin precipitate in agarose gels. This precipitate induced in rabbits a potent antiserum specific for the gonococcal antigen which reacted with WGA. Immune electron microscopy on whole gonococci showed that this antiserum reacted with outer membrane vesicles. Lipopolysaccharide (LPS) purified by phenol-water extraction of whole gonococci reacted with WGA and also with the antiserum to the WGA-antigen complex. This indicated that the antiserum was specific for antigen(s) of the outer membrane complex.

Intracellular killing of Candida albicans by human polymorphonuclear leucocytes: comparison of three methods of assessment.

Three different methods, [3H]uridine uptake, viable count and 51Cr-release were used to assess the intracellular survival of a strain of Candida albicans, 19321, which was lethal for mice injected intravenously. Intracellular survival 1 h after ingestion ranged from 50 to 80% depending on the method employed and the detergent used to lyse the phagocytes. Inhibition of uridine uptake by detergents used to lyse the phagocytes led to difficulty in assessment of intracellular killing by this method.

The role of RNA segment 1 in an in vitro host restriction occurring in an avian influenza virus mutant.

A temperature sensitive mutant, ts C47, derived from A/FPV/Rostock/34 and with a ts mutation in RNA segment 8, fails to form plaques in MDCK cells. From data obtained with reassortant viruses using the human influenza isolate A/FM/1/47 it was apparent that more than one mutation contributed to the temperature-sensitive (ts) and host range (hr) phenotypes of ts C47, and the phenotype of reassortants containing RNA segment 1 from A/FM/1/47 indicated that this segment was involved. A single nucleotide substitution at nucleotide 1961, resulting in valine instead of methionine in the predicted amino acid sequence of polypeptide PB2, was found in RNA segment 1 of ts C47, but this mutation did not segregate with the attenuated phenotype on gene reassortment. The following conclusions are drawn: (a) that ts C47 has at least two mutations in addition to that already known to exist in RNA segment 8, one of which (that in RNA segment 1) does not contribute to the observed ts hr phenotypes and (b) that the hr phenotype can be suppressed by substitution of RNA segment 1 by that of another strain.

Capped mRNAs may stimulate the influenza virion polymerase by allosteric modulation.

Analogues of the mRNA 5'-terminal methyl cap structure were found to stimulate the influenza virion RNA-dependent RNA polymerase. The single nucleotide analogue m7GMP was incorporated into RNA during transcription in vitro, and the stimulatory effect was not additive with the primer ApG, suggesting that m7GMP stimulates the virion polymerase by priming virus-specific mRNA synthesis, as has been shown for ApG. By contrast, stimulation by m7G(5')ppp(5')m6AM2-O was additive with that by ApG, and we could not demonstrate incorporation of the similar analogue m7G(5')ppp(5')Am2-O into RNA during transcription. We propose that these dinucleotide cap analogues stimulate the virion polymerase by allosteric modulation, independent of priming. This stimulation can be abolished by mutation, without loss of other activities associated with the cap-dependent endonuclease.

Novel polypeptides encoded by influenza virus subgenomic (DI type) virion RNAs.

We have isolated a ts mutant of influenza A/FPV/Rostock/34 that induces the synthesis of a novel small polypeptide in infected cells. This polypeptide is encoded by a subgenomic virion RNA derived from RNA segment 3, apparently by internal deletion. A second polypeptide, similarly derived from RNA segment 1, was found only after in vitro translation of infected cell RNA. The subgenomic vRNAs we describe are probably similar to those found in influenza DI virus preparations. The possible role of 'subgenomic' polypeptides in DI virus-mediated interference is discussed.

The critical cut-off temperature of avian influenza viruses.

We have measured the pathogenicity for 6-week-old chicks of infection by H7 avian influenza viruses. One virus, strain S3 from A/FPV/Rostock/34(H7N1) showed a temperature sensitive phenotype at 41.5 degrees C and reduced pathogenicity. By analysis of reassortants made between virus S3 and A/FPV/Dobson/27(H7N7), a fully pathogenic virus, two conclusions arise. (1) The critical cut-off temperature for avian influenza virus in 6-week-old chicks is 41.5 degrees. (2) RNA segment 1 of virus S3 is responsible for the lack of pathogenicity in reassortant viruses. Nucleotide sequencing of RNA segment 1 from S3 and its parent, A/FPV/Rostock/34 has revealed a single mutation at nucleotide 1561. This results in a substitution of isoleucine for leucine at amino acid position 512 in the cap-binding protein, PB2.

Characterisation of the influenza virus associated protein kinase and its resemblance to casein kinase II.

The protein kinase activity associated with purified influenza virus has been examined. By use of a radiolabelled photoreactive ATP analogue (3'-O-(4-benzoyl) benzoyl adenosine triphosphate) a 47 kD polypeptide has been identified that binds ATP. A comparison of the sensitivity of the kinase activity and the 47 kDa polypeptide labelling to inhibitors indicate that the 47 kDa polypeptide is likely to represent the major protein kinase activity of the virus. The virus associated protein kinase phosphorylates the synthetic peptide RREEETEEE, a peptide substrate for casein kinase II. Antiserum directed against casein kinase II revealed a positive signal in immunoblots of purified virus. We propose that the major protein kinase activity associated with purified virus preparations is host cell casein kinase II.

The intracellular phosphorylation of (-)-2'-deoxy-3'-thiacytidine (3TC) and the incorporation of 3TC 5'-monophosphate into DNA by HIV-1 reverse transcriptase and human DNA polymerase gamma.

(-)-2'-deoxy-3'-thiacytidine (3TC) has been shown to be a potent, selective inhibitor of HIV replication in vitro, which requires phosphorylation to its 5'-triphosphate for antiviral activity. The intracellular concentration of 3TC 5'-triphosphate in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) shows a linear dependence on the extracellular concentration of 3TC up to an extracellular 3TC concentration of 10 microM. At this extracellular concentration of 3TC, the resulting intracellular concentration of 3TC 5'-triphosphate is 5 microM. This value is similar to the inhibition constant (Ki) values for the competitive inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and human DNA polymerases (10-16 microM) by 3TC 5'-triphosphate. Since the concentration of 3TC producing 90% inhibition (IC90) of HIV replication in PBLs has been reported to be 76 nM, the antiviral activity of 3TC requires intracellular concentrations of 3TC 5'-triphosphate, which would result in very little inhibition of reverse transcriptase if its sole mode of action was competitive inhibition. This apparent discrepency may be explained by the ability of 3TC 5'-triphosphate to act as a substrate for reverse transcriptase. Primer extension assays have shown that 3TC 5'-triphosphate is a substrate for HIV-1 reverse transcriptase and DNA polymerase gamma, resulting in the incorporation of 3TC 5'-monophosphate into DNA. In the case of DNA polymerase gamma, the product of this reaction (i.e. double-stranded DNA with 3TC 5'-monophosphate incorporated at the 3'-terminus of the primer strand) is also a substrate for the 3'-5' exonuclease activity of this enzyme. This may explain the low levels of mitochondrial toxicity observed with 3TC.

Cellular metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine.

The metabolism of (-) enantiomeric 2'-deoxy-3'-thiacytidine (3TC) was examined in human immunodeficiency virus type 1 (HIV-1)-infected and mock-infected human cells. 3TC 5'-triphosphate levels accumulated comparably in HIV-1-infected and mock-infected phytohaemagglutinin-stimulated peripheral blood lymphocytes (PBL) and reached 40% or more of total intracellular 3TC metabolites after 4 hr. The rate of decay of 3TC triphosphate in HIV-1-infected and mock-infected PBL measured as a half-life (T1/2) ranged from 10.5 to 15.5 hr. 3TC did not significantly affect metabolism of deoxynucleotides in the U937 cell line, and was shown to be resistant to the action of human platelet pyrimidine nucleoside phosphorylase.

Immune response to Treponema pertenue and Treponema pallidum Nichols in patients with yaws.

Human sera from African patients with acute yaws were analysed by Western blot (WB) against antigens of Treponema pallidum Nichols and two Treponema pertenue isolates. The Western blot patterns were remarkably similar from one patient to another, and strains of both subspecies exhibited exactly the same banding pattern. Sera from yaws patients failed to detect at least one antigen in T. pertenue which was absent from T. pallidum.

Interaction of spirochetes with the host.

The success of an invading organism must depend on several cytoplasmic, surface-associated and secreted factors. The technical difficulties in handling pathogenic spirochetes like Treponema pallidum and Borrelia burgdorferi have made it difficult to define specific factors involved in entry and long-term survival. The problem of defining virulence factors has been attacked by several strategies: T. pallidum secretes a number of immunogenic low molecular mass proteins. The most predominant are of molecular weight 15.5 and 22 kDa. Preliminary data suggest that antibodies against these proteins induce protective immunity in rabbits experimentally infected with T. pallidum. Many potentially important surface-associated antigens of T. pallidum have now been cloned and characterized. Two of these, TpD and TpE, are lipoproteins which exhibit characteristic size heterogeneity. The apparent molecular weight of TpE from T. pallidum and T. pertenue are different. The clinical symptoms in syphilis and yaws are very different, but sequence analysis of TpE has shown that the TpE proteins are indeed very similar in the two strains. This observation makes it unlikely that heterogeneity of TpE can account for the different clinical symptoms of syphilis and yaws. Sequence data for another newly sequenced surface-associated antigen of T. pallidum (molecular weight 41 kDa) indicate that this protein is involved in glucose transport and chemotaxis/motility. Intracellular factors like the molecular chaperonin GroEL have been documented both in treponemes and borreliae. This stress protein is involved in cellular repair processes and folding/assembly of protein subunits. Indirect evidence suggests that GroEL affects the ability of spirochetes to survive in the stressful environment of the infected host. Several lines of evidence suggest that the Osp proteins of Borrelia are important for host/parasite interaction. Further support for this idea has come from studies of a series of monoclonal antibodies against OspA. A monoclonal antibody against OspA (9B3D) is able to block attachment of B. burgdorferi to a cell monolayer. Borrelia loses infectivity after several passages in vitro. The loss of pathogenicity is associated with loss of specific plasmids and proteins. One of the low-passage-associated proteins (Lap30) has been cloned and sequenced. Lap30 is a lipoprotein encoded by a 38-kb plasmid, not present in high passage B. burgdorferi. Aberrant immunological processes induced by the lipopolysaccharide component of Treponema hyodysenteriae could explain the dramatic intestinal lesions in swine dysenteriae. But analysis by TLC reveals that the LPS of this treponeme is different from classical Salmonella LPS.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization and gene sequencing of a 19-kDa periplasmic protein of Campylobacter jejuni/coli.

In order to study a 19-kDa protein (p19) of Campylobacter jejuni, we purified this protein to homogeneity from C. jejuni strain 81,176 by anion exchange chromatography. The molecular weight of the native protein is 19,000 daltons. P19 was found to be acidic with an isoelectric point of 4.8 and was located in the periplasmic space of the bacteria. The 20 N-terminal amino acids were sequenced and no significant similarities with known proteins were shown. A monoclonal antibody showed that p19 is conserved in the 2 species C. jejuni and C. coli. Analysis of sera from 23 patients with a Campylobacter-related infection indicated that p19 is not immunogenic during natural infection in man. The gene encoding p19 was cloned and no strong homologies with known sequences were identified. The preparation of a knockout mutant in p19 will enable the investigation of the function of this cell wall component of Campylobacter.

Genetic approaches to cell biology and metabolism of spirochetes.

Genetic analysis and methodology have only comparatively recently been applied to the study of spirochetes. Although genetic transfer procedures for spirochetes are not widely available, there are several examples of progress in genetic analysis of spirochetes by other approaches. Some examples of these approaches are the following. 1) Genes for synthetic pathways in Treponema and Leptospira have been cloned by complementation of Escherichia coli serving as plasmid hosts. 2) The OspA protein of Borrelia burgdorferi has been overexpressed in E. coli without the signal peptide; the recombinant product has been suitable for circular dichroism as well as other biochemical analyses. 3) The heat shock proteins of B. burgdorferi are homologous to heat shock proteins of E. coli. 4) Enzyme activity profiles of B. burgdorferi and other spirochetes show strain heterogeneity and also indicate which biosynthetic and enzymatic activities are conserved within different spirochetes. 5) The gene organization of rRNA genes have revealed differences between spirochetes and other types of bacteria.

Inhibition of the growth of influenza viruses in vitro by 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid.

The sialidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N- acetylneuraminic acid was tested for growth inhibitory effects against a panel of avian influenza A viruses encompassing all nine neuraminidase subtypes. Growth in tissue culture of viruses from each subtype was inhibited by this compound at concentrations within a range previously found effective against human N1 and N2 viruses. This compound may prove a selective agent for the treatment (and prevention) of influenza virus infections.

4-Guanidino-Neu5Ac2en fails to protect chickens from infection with highly pathogenic avian influenza virus.

The effectiveness of the novel sialidase inhibitor 4-guanidino-Neu5Ac2en, which is highly effective in mouse and ferret models of influenza virus infection (von Itzstein et al. (1993) Nature 363, 418-423), has been assessed as a prophylactic agent in the prevention of infection of chickens with highly pathogenic avian influenza viruses. At best a small delay in the onset of pyrexia and death was observed with one strain of fowl plague virus, but not with two other strains. These results demonstrate that a locally acting drug may be ineffective if virus can escape from the site of inoculation and replicate elsewhere.