Antipneumococcal activity of neuraminidase inhibiting artocarpin.

Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase (NA) is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases (NAs) are structurally related to viral NA and susceptible to oseltamivir, an inhibitor designed to target viral NA. This prompted us to evaluate the antipneumococcal potential of two NA inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC(50) value) of the tested compounds towards pneumococcal NAs. The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA NA. Unlike oseltamivir, which competes with the natural substrate of NA, artocarpin exhibits a mixed-type inhibition with a Ki value of 9.70 µM. Remarkably, artocarpin was the only NA inhibitor (NAI) for which an inhibitory effect on pneumococcal growth (MIC: 0.99-5.75 µM) and biofilm formation (MBIC: 1.15-2.97 µM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by a factor of >1000, without obvious harm to lung epithelial cells. This renders artocarpin a promising natural product for further investigations.

Dynamic behaviour of selected PET tracers in embryonated chicken eggs

La tomografía por emisión de positrones/tomografía computarizada (PET/TC) es una técnica establecida en la investigación preclínica de enfermedades en modelos de animales peque˜nos y en el diagnóstico clínico de cáncer. Esta técnica combina la información funcional del biomarcador emisor de positrones con los datos anatómicos de la imagen TC, permitiendo de este modo la investigación in vivo de los procesos biológicos en 4 dimensiones. Recientemente, el crecimiento de los huesos de los embriones de pollo se ha podido monitorizar usando PET/TC con 18F fluoruro como trazador óseo. Estamos interesados en la investigación de otros trazadores PET/TC en embriones de pollo como sistema de modelo in vivo. Para ello, evaluamos varios radiotrazadores usados habitualmente en pruebas clínicas o sintetizamos aquellos que no estaban disponibles comercialmente. Utilizando un microPET/TC de animales peque˜nos, evaluamos las características de 18F, 68Ga y 64Cu cloruro en huevos con sondaje. La 2-Deoxy-2[18F]fluorodeoxiglucosa (18F FDG) estaba absorbida en los sitios de crecimiento de los huesos. El 64Cu cloruro y un anticuerpo de unión a fibrillas de amiloide marcado con 68Ga se acumularon en el hígado, mientras que el 68Ga ligado a un conjugado de desferroxamina disminuyó con el tiempo en ese mismo órgano. Los resultados indican que estos biomarcadores pueden ser utilizados para la monitorización de procesos biológicos en huevos de pollo como modelo animal (AU)

Positron emission tomography/computer tomography (PET/CT) is an established method in preclinical research in small animal disease models and the clinical diagnosis of cancer. It combines functional information of the positron-emitting biomarker with the anatomical data obtained from the CT image. Thus, it allows for 4D in vivo investigation of biological processes. Recently, PET/CT was used to monitor bone growth of chicken embryos using 18F-fluoride as a bone-seeking tracer. We are interested in investigating the adequacy of additional PET/CT tracers in chicken embryos as an in vivo model system. For this reason, we evaluated several positron emitting compounds typically used in clinical tests or if these were not commercially available, we synthesised them. We studied the properties of these 18F- and 68Ga-labelled tracers and of 64Cu-chloride in catheterised eggs via small animal microPET/CT. 2-Deoxy-2-[18F]fluoroglucose ([18F]FDG) was primarily absorbed at the sites of bone growth. 64Cu chloride and a 68Ga-labelled amyloid-fibril-binding antibody accumulated in the liver, while the 68Ga-albumin desferrioxamine conjugate signal in liver decreased over time. These results indicate that these biomarkers can potentially be used for the monitoring of biological processes in chicken eggs as an animal model (AU)

Dynamic behaviour of selected PET tracers in embryonated chicken eggs.

Positron emission tomography/computer tomography (PET/CT) is an established method in preclinical research in small animal disease models and the clinical diagnosis of cancer. It combines functional information of the positron-emitting biomarker with the anatomical data obtained from the CT image. Thus, it allows for 4D in vivo investigation of biological processes. Recently, PET/CT was used to monitor bone growth of chicken embryos using (18)F-fluoride as a bone-seeking tracer. We are interested in investigating the adequacy of additional PET/CT tracers in chicken embryos as an in vivo model system. For this reason, we evaluated several positron emitting compounds typically used in clinical tests or if these were not commercially available, we synthesised them. We studied the properties of these (18)F- and (68)Ga-labelled tracers and of (64)Cu-chloride in catheterised eggs via small animal microPET/CT. 2-Deoxy-2-[(18)F]fluoroglucose ([(18)F]FDG) was primarily absorbed at the sites of bone growth. (64)Cu chloride and a (68)Ga-labelled amyloid-fibril-binding antibody accumulated in the liver, while the (68)Ga-albumin desferrioxamine conjugate signal in liver decreased over time. These results indicate that these biomarkers can potentially be used for the monitoring of biological processes in chicken eggs as an animal model.

Identification and characterization of toxin-antitoxin systems in strains of Lactobacillus rhamnosus isolated from humans.

The toxin-antitoxin gene systems (TASs) are present in the genomes of the overwhelming majority of bacteria and archaea. These systems are involved in various cellular regulatory processes (including stress response), and have not been previously investigated in Lactobacilli. We identified 6 putative TASs with toxins belonging to the MazE and RelE superfamilies (PemK1-А1Lrh, PemK2-А2Lrh, PemK3-RelB2Lrh, RelE1Lrh, RelB3-RelE3Lrh, and YefM-YoeBLrh) in the genomes of annotated strains of Lactobacillus rhamnosus. PCR analyses revealed that all systems were found in the genomes of 15 strains of L. rhamnosus isolated from humans in central Russia. These strains were highly heterogeneous with respect to the presence of TASs, as well as their nucleotide and amino acid sequences. In three cases, the relE1 genes contained IS3 elements. TAS heterogeneity may be used to reveal inter-genus differences between strains. Cloning of the toxin genes of 3 TASs inhibited Escherichia coli growth, thus confirming their functionality. Cell growth arrest caused by expression of the toxin genes could be reverted by the expression of a cognate antitoxins. Transcription of toxin-antitoxin loci in L. rhamnosus was shown by RT-PCR.

Computer controlled 68Ga milking and concentration system.

Here we describe an automated (68)Ge/(68)Ga generator milking and concentration procedure. It is especially suited for reproducible preparation of (68)Ga in volumes of approximately 200 microl with optimum activity for subsequent labelling of small amounts of peptides or proteins ideal for microPET applications.

Phenotypic, genetic and genomic consequences of natural and synthetic polyploidization of Nicotiana attenuata and Nicotiana obtusifolia.

BACKGROUND AND METHODS: Polyploidy results in genetic turmoil, much of which is associated with new phenotypes that result in speciation. Five independent lines of synthetic allotetraploid N. x obtusiata (N x o) were created from crosses between the diploid N. attenuata (Na) (male) and N. obtusifolia (No) (female) and the autotetraploids of Na (NaT) and No (NoT) were synthesized. Their genetic, genomic and phenotypic changes were then compared with those of the parental diploid species (Na and No) as well as to the natural allotetraploids, N. quadrivalvis (Nq) and N. clevelandii (Nc), which formed 1 million years ago from crosses between ancient Na and No. KEY RESULTS: DNA fingerprinting profiles (by UP-PCR) revealed that the five N x o lines shared similar but not identical profiles. Both synthetic and natural polyploidy showed a dosage effect on genome size (as measured in seeds); however, only Nq was associated with a genome upsizing. Phenotypic analysis revealed that at the cellular level, N x o lines had phenotypes intermediate of the parental phenotypes. Both allo- and autotetraploidization had a dosage effect on seed and dry biomass (except for NaT), but not on stalk height at first flower. Nc showed paternal (Na) cellular phenotypes but inherited maternal (No) biomass and seed mass, whereas Nq showed maternal (No) cellular phenotypes but inherited paternal (Na) biomass and seed mass patterns. Principal component analysis grouped Nq with N x o lines, due to similar seed mass, stalk height and genome size. These traits separated Nc, No and Na from Nq and N x o lines, whereas biomass distinguished Na from N x o and Nq lines, and NaT clustered closer to Nq and N x o lines than to Na. CONCLUSIONS: Both allo- and autotetraploidy induce considerable morphological, genetic and genomic changes, many of which are retained by at least one of the natural polyploids. It is proposed that both natural and synthetic polyploids are well suited for studying the evolution of adaptive responses.

Fabrication and evaluation of a sequence-specific oligonucleotide miniarray for molecular genotyping.

PURPOSE: Molecular genotyping relies on the identification of specific microbial DNA sequences. Accurate genotyping not only requires discrimination between low- and high-risk pathogens for effective diagnosis or disease management but also requires the identity of the specific strain or type of the microbe involved in pathogenesis. The majority of these assays require DNA amplification followed by genome identification either through sequencing or hybridization to specific oligonucleotide probes. We evaluated the use of a DNA microchip assay as a simple and easy-to-use procedure for genotyping. METHODS: Various methodological parameters were optimized for single-base mismatch discrimination on a DNA microarray. The fabrication procedures involved substrate chemistry for immobilization. The effect of various buffers and features associated with oligonucleotide sequences were standardized. The assay was evaluated on a low-density genotyping chip containing the sequences of various (Human Papilloma Virus) HPV subtypes. RESULTS: The specific subtype was identified with high specificity by hybridization in miniaturized condition. CONCLUSIONS: The DNA microchip provides a rapid and cost-effective genotyping procedure for microbial organisms and can be implemented easily in any laboratory.

The apoptotic capability of coxsackievirus B3 is influenced by the efficient interaction between the capsid protein VP2 and the proapoptotic host protein Siva.

Infections with coxsackievirus B3 (CVB3) are common causes of myocarditis in humans. One detail of CVB3-induced pathogenesis is apoptosis. The interaction between the capsid protein VP2 of the myocardial virus variant CVB3H3 and the proapoptotic host cell protein Siva has recently been observed. In order to characterize the interaction between both proteins more precisely, the binding activity of the CVB3H3 VP2 to Siva was compared to that of the mutant virus CVB3H310A1 VP2. We found that the asparagine at position 165 in VP2 is essential for a stable interaction with Siva influencing also the induction of apoptosis, viral spread, and inflammatory responses in vivo. Furthermore, the specific binding site of Siva to VP2 is located at amino acid positions 118-136. Together, these results show that the interaction between VP2 of CVB3H3 and Siva is a highly specific process involving distinct amino acids on both proteins that most likely influence the outcome of CVB3-caused disease.

Functional analysis of genes from Streptomyces griseus involved in the synthesis of isorenieratene, a carotenoid with aromatic end groups, revealed a novel type of carotenoid desaturase.

The biosynthesis of the aromatic carotene isorenieratene is restricted to green photosynthetic bacteria and a few actinomycetes. Among them Streptomyces griseus has been used to study the genes involved in this pathway. Five genes out of seven of two adjacent operons in one cluster could be identified to be sufficient for the synthesis of isorenieratene. Stepwise deletions of these genes demonstrated their participation in phytoene synthesis, phytoene desaturation and lycopene cyclization. The novel gene crtU was assigned to encode a unique desaturase responsible for the conversion of beta-carotene via beta-isorenieratene to isorenieratene by a desaturation/methyltransferation mechanism. Sequence analysis of crtU revealed two conserved regions, one at the N-terminus and the other at the C-terminus of the protein which is universal to different types of carotene desaturases. In addition, the sequence comprises a motif typically found in methyltransferases. The deletion of the two remaining genes of the cluster left the carotenoid biosynthetic pathway unaffected.

Identification of a gene encoding the pyruvate decarboxylase gene regulator CaPdc2p from Candida albicans.

In a screen for Candida albicans genes encoding transactivating proteins, a pyruvate decarboxylase (EC 4.1.1.1.) regulator gene was isolated. An open reading frame (ORF) of 2511 bp was identified encoding a predicted protein of 836 amino acids with a molecular weight of 94.4 kDa. The protein showed glutamine- and proline-rich stretches typical for transcriptional activators. The amino acid sequence comparisons between CaPdc2p of C. albicans and both Pdc2p of Saccharomyces cerevisiae and Rag3p of Kluyveromyces lactis, revealed similarities of 40% and 39%, respectively. The CaPDC2 gene was localized on chromosome 1. Southern blot analysis indicated that CaPDC2 might be a single copy gene. The growth defect of a S. cerevisiae pdc2 delta mutant on glucose was compensated by transformation of the C. albicans CaPDC2 gene.

UV-Laser induced protein/DNA crosslinking reveals sequence variations of DNA elements bound by c-Jun in vivo.

Many proteins involved in the modulation of gene expression exert their function through direct interaction with DNA. The sequence specificity of these interactions provides the basis for many regulatory mechanisms. The sites that are utilized by a transcription factor are usually analyzed using in vitro binding studies. To detect true in vivo binding sites we developed a method, presented here, that allows construction of recognition element DNA (reDNA) libraries which represent in vivo binding sites plus flanking sequences. reDNA libraries can be constructed for any well-characterized transcription factor. Here we used this method for an in vivo study of genomic DNA elements that interact with the transcription factor c-Jun in rat cerebellum.

Mg-chelatase of tobacco: the role of the subunit CHL D in the chelation step of protoporphyrin IX.

The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein-protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.

Analysis of a 26,756 bp segment from the left arm of yeast chromosome IV.

The nucleotide sequence of a 26.7 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome IV is presented. An analysis of this segment revealed 11 open reading frames (ORFs) longer than 300 bp and one split gene. These ORFs include the genes encoding the large subunit of RNA polymerase II, the biotin apo-protein ligase, an ADP-ribosylation factor (ARF 2), the 'L35'-ribosomal protein, a rho GDP dissociation factor, and the sequence encoding the protein phosphatase 2A. Further sequence analysis revealed a short ORF encoding the ribosomal protein YL41B, an intron in a 5' untranslated region and an extended homology with another cosmid (X83276) located on the same chromosome. The potential biological relevance of these findings is discussed.

DNA binding and methyl transfer catalysed by mouse DNA methyltransferase.

By using a purified fraction of mouse DNA methyltransferase we have shown, by gel-retardation analysis, that the enzyme forms a low-affinity complex preferentially with hemimethylated DNA; the complexes formed with unmethylated or with fully methylated DNA are of even lower affinity, and only very weak interaction occurs with DNA lacking CG dinucleotides. Interaction is inhibited by N-ethylmaleimide. Methyl transfer from S-adenosyl-methionine is associated with the release of the fully methylated product from the complex. Complexes formed with the intact enzyme are extremely large, but limited trypsin treatment allows a major complex to enter the gel. DNA binding is not inhibited by this limited proteolysis of the native enzyme.

Binding of histone H1e-c variants to CpG-rich DNA correlates with the inhibitory effect on enzymic DNA methylation.

Within the H1 histone family, only some fractions enriched in the H1e-c variants are effective in causing a marked inhibition, in vitro, of enzymic DNA methylation and, in gel retardation and Southwestern blot experiments, in binding double-stranded (ds) CpG-rich oligonucleotides. Both the 6-CpG ds-oligonucleotide and the DNA purified from chromatin fractions enriched in 'CpG islands' are good competitors for the binding of H1e-c to 6-meCpG ds-oligonucleotide. Because of their ability to bind any DNA sequence and to suppress the enzymic methylation in any sequence containing CpG dinucleotides, these particular H1 variants could play some role in maintaining linker DNA at low methylation levels and even in preserving the unmethylated state of the CpG-rich islands which characterize the promoter regions of housekeeping genes.

Approaches to characterize protein-DNA interactions in vivo.

With the availability of direct genomic footprinting techniques the study of native genomes has been greatly facilitated. This review provides an overview of the techniques involved and gives also a description of the mode of action of different DNA modifying agents which can be used for such methods. These include exonuclease III, deoxyribonuclease, DNase I, micrococcal nuclease, dimethyl sulfate, diethyl sulfate, ethyl methanesulphonate, ethylnitrosourea, diethylpyrocarbonate, bromoacetaldehyde, potassium permanganate, osmium tetroxide, methidiumpropyl-EDTA-iron(II), formaldehyde, psoralen, 1,10 phenanthroline-copper and UV light. We also describe the limitations of the currently existing techniques and give some potential developments.

Ultramicrodetection of proteins in polyacrylamide gels.

Here we report the development of a highly sensitive procedure to detect proteins within separation matrices which should facilitate the characterization of rare proteins. The procedure is based on photochemical reactions where very low amounts of silver are deposited around proteins and in a series of steps are converted to silver sulfide. When this conversion is carried out in the presence of [35S]thiourea the resulting radioactive silver sulfide allows detection down to femtogram quantities of protein. In this work we applied the above principle to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, thus not influencing physical and chemical parameters which are important for separation. This procedure should find application in any technique where detection of very low or limited amounts of proteins are required.

Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site.

A negative regulating protein (MDBP-2) from rooster liver nuclear extracts binds preferentially to a methylated promoter region 5'TTCACCTTmCGCTATGAGGGGGATCATACTGG3' of the avian vitellogenin II gene (Nucleic Acids Res. 19, 1029-1034, 1991). Treatment of adult and immature roosters with estradiol results in a 90% decrease in the binding activity of MDBP-2 within three days. This corresponds to the level found in egg laying hens. The decrease in the binding activity of MDBP-2 precedes the onset of vitellogenin gene transcription. At the same time, there is a two-fold increase in the binding activity of NHP-1 (tested with the same oligonucleotide as for MDBP-2), a protein thought to be involved in the active demethylation of DNA. The methylated oligonucleotide binds either MDBP-2 or NHP-1 and there is no complex formation between the two proteins and DNA. Estradiol treatment does not change the equilibrium binding constant of MDBP-2 which is about 10(-9)M for the methylated oligonucleotide. The early kinetics of demethylation of the mCpG pair in the binding site of MDBP-2 was studied by means of genomic sequencing. A low level of demethylation of mCpG starts gradually on both DNA strands already 4 hours after estradiol treatment during the lag phase of vitellogenin mRNA synthesis. It is concluded that the lowering of the binding activity of MDBP-2 may have a stronger effect on the derepression of the gene than the slow demethylation of MDBP-2 DNA binding site. The role of the methylated CpG is to assure a high binding affinity of the repressor to DNA.