Role of the cgtA gene function in DNA replication of extrachromosomal elements in Escherichia coli

The cgtA gene codes for a common GTP-binding protein whose homologues were found in all prokaryotic and eukaryotic organisms investigated so far. Although cgtA is an essential gene in most bacterial species, its precise functions in the regulation of cellular processes are largely unknown. In Escherichia coli, dysfunction or overexpression of the cgtA gene causes problems in various chromosomal functions, like synchronization of DNA replication initiation and partitioning of daughter chromosomes after a replication round. It is not know how the cgtA gene product regulates these processes. Here we investigated effects of cgtA dysfunction on replication of plasmid and phage replicons. We found that replication of some plasmids (e.g., ColE1-like) is not affected in the cgtA mutant. On the other hand, dysfunction of the cgtA gene caused a strong inhibition of lambda plasmid DNA replication. Bacteriophage lambda development was severely impaired in the cgtA mutant. Replication of other plasmid replicons (derivatives of F, R1, R6K, and RK2) was influenced by the cgtA mutation moderately. It seems that DNA synthesis per se is not affected by CgtA, and that this protein might control replication initiation indirectly, by regulation of function(s) or production of one or more replication factors. In fact, we found that level of the host-encoded replication protein DnaA is significantly decreased in the cgtA mutant. This indicates that CgtA is involved in the regulation of dnaA gene expression.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)⁶]- and [D-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH₂)⁶-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Shell Beds as paleoecological puzzles: A case study from the Upper Permian of the Paraná Basin, Brazil

Summary Microstratigraphic, sedimentological, and taphonomic features of the Ferraz Shell Bed, from the Upper Permian (Kazanian-Tatarian?) Corumbataí Formation of Rio Claro Region (the Paraná Basin, Brazil), indicate that the bed consists of four distinct microstratigraphic units. They include, from bottom to top, a lag concentration (Unit 1), a partly reworked storm deposit (Unit 2), a rapidly deposited sandstone unit with three thin horizons recording episodes of reworking (Unit 3), and a shell-rich horizon generated by reworking/winnowing that was subsequently buried by storm-induced obrution deposit (Unit 4). The bioclasts of the Ferraz Shell Bed represent exclusively bivalve mollusks.Pinzonellaillusa andTerraia aequilateralis are the dominant species. Taphonomic analysis indicates that mollusks are heavily time-averaged (except for some parts of Unit 3). Moreover, different species are time-averaged to a different degree (disharmonious time-averaging). The units differ statistically from one another in their taxonomic and ecological composition, in their taphonomic pattern, and in the size-frequency distributions of the two most common species. Other Permian shell beds of the Paraná Basin are simílar to the Ferraz Shell Bed in their faunal composition (they typically contain similar sets of 5 to 10 bivalve species) and in their taphonomic, sedimentologic, and microstratigraphic characteristics. However, rare shell beds that include 2–3 species only and are dominated by articulated shells preserved in life position also occur. Diversity levels in the Permian benthic associations of the Paraná Basin were very low, with the point diversity of 2–3 species and with the within-habitat and basin-wide (alpha and gamma) diversities of 10 species, at most. The Paraná Basin benthic communities may have thus been analogous to low-diversity bivalve-dominated associations of the present-day Baltic Sea. The ‘Ferraz-type’ shell beds of the Paraná Basin represent genetically complex and highly heterogeneous sources of paleontological data. They are cumulative records of spectra of benthic ecosystems time-averaged over long periods of time (10²–10⁴ years judging from actualistic research). Detailed biostratinomic reconstructions of shell beds can not only offer useful insights into their depositional histories, but may also allow paleoecologists to optimize their sampling designs, and consequently, refine paleoecological and paleoenvironmental interpretations.     

The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.     

Study of a major intermediate in the oxidative folding of leech carboxypeptidase inhibitor: contribution of the fourth disulfide bond

The oxidative folding pathway of leech carboxypeptidase inhibitor (LCI; four disulfide bonds) proceeds through the formation of two major intermediates (III-A and III-B) that contain three native disulfide bonds and act as strong kinetic traps in the folding process. The III-B intermediate lacks the Cys19-Cys43 disulfide bond that links the beta-sheet core with the alpha-helix in wild-type LCI. Here, an analog of this intermediate was constructed by replacing Cys19 and Cys43 with alanine residues. Its oxidative folding follows a rapid sequential flow through one, two, and three disulfide species to reach the native form; the low accumulation of two disulfide intermediates and three disulfide (scrambled) isomers accounts for a highly efficient reaction. The three-dimensional structure of this analog, alone and in complex with carboxypeptidase A (CPA), was determined by X-ray crystallography at 2.2A resolution. Its overall structure is very similar to that of wild-type LCI, although the residues in the region adjacent to the mutation sites show an increased flexibility, which is strongly reduced upon binding to CPA. The structure of the complex also demonstrates that the analog and the wild-type LCI bind to the enzyme in the same manner, as expected by their inhibitory capabilities, which were similar for all enzymes tested. Equilibrium unfolding experiments showed that this mutant is destabilized by approximately 1.5 kcal mol(-1) (40%) relative to the wild-type protein. Together, the data indicate that the fourth disulfide bond provides LCI with both high stability and structural specificity.     

Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.     

Prenatal Stress Enhances Excitatory Synaptic Transmission and Impairs Long-Term Potentiation in the Frontal Cortex of Adult Offspring Rats

The effects of prenatal stress procedure were investigated in 3 months old male rats. Prenatally stressed rats showed depressive-like behavior in the forced swim test, including increased immobility, decreased mobility and decreased climbing. In ex vivo frontal cortex slices originating from prenatally stressed animals, the amplitude of extracellular field potentials (FPs) recorded in cortical layer II/III was larger, and the mean amplitude ratio of pharmacologically-isolated NMDA to the AMPA/kainate component of the field potential—smaller than in control preparations. Prenatal stress also resulted in a reduced magnitude of long-term potentiation (LTP). These effects were accompanied by an increase in the mean frequency, but not the mean amplitude, of spontaneous excitatory postsynaptic currents (sEPSCs) in layer II/III pyramidal neurons. These data demonstrate that stress during pregnancy may lead not only to behavioral disturbances, but also impairs the glutamatergic transmission and long-term synaptic plasticity in the frontal cortex of the adult offspring.