Molecular Mechanism of Heat Shock-Provoked Disassembly of the Coliphage λ Replication Complex

We have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the λO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of λ plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from λ DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to λcro⁺, in λcro⁻ plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the λcrots plasmid-harboring cells, a Cro repressor-independent control of λ DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to λ DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the λcro⁻ plasmid replication control.     

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.     

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.     

Effect of Brood Age on Nestling Diet and Prey Composition in a Hedgerow Specialist Bird,the Barred Warbler Sylvia nisoria

The composition and quality of food provided to nestling birds influence their growth and development and offers key insight into the ecological requirements of birds. One bird species whose feeding ecology is poorly understood is the Barred Warbler (Sylvia nisoria), which utilizes semi-natural shrubby vegetation in agroecosystems. Because Barred Warbler nestlings vary greatly in body mass we hypothesised that diet and prey properties (size, diversity, taxonomic composition, and chitin content and resulting body hardness and digestibility) would differ as the nestlings aged. We quantified the diet based on faecal analysis, sampling faecal sacs from the nestlings pooled into three age classes: 2-3 days old, 4-6 d old, and 7-9 d old. Nestlings were provided a wide diversity of food and a strong relationship existed between food characteristics and nestling age. The youngest nestlings (2-3 d old) had the lowest values of each dietary characteristic (diversity, number and total biomass of prey, and individual prey weight), that were significantly lower than the oldest nestlings (7-9 d old). Nestlings aged 4-6 d exhibited intermediate dietary characteristics. Differences in dietary composition of the six major food types showed marked differences between the individual broods and age categories. Percentages of the number and biomass of soft-bodied prey were highest in the diet of 2-3 d and 4-6 d old nestlings, and decreased with increasing age, whereas the opposite trend was observed in the percentage of intermediately and heavily chitinised prey. Parent Barred Warblers probably preferentially select soft-bodied prey for the youngest nestlings, and satisfy the greater energy demands of the older ones by providing them with a greater variety of prey containing more chitin, as well as plant food. The provisioning of less-readily digestible prey to older nestlings suggests that as the quality of food decreases the quantity increases, implying that the youngest nestlings may be physiologically limited as regards their ability to digest more heavily chitinised prey.     

The Molecular Mechanism of Amyloid β42 Peptide Toxicity: The Role of Sphingosine Kinase-1 and Mitochondrial Sirtuins

Our study focused on the relationship between amyloid β 1–42 (Aβ), sphingosine kinases (SphKs) and mitochondrial sirtuins in regulating cell fate. SphK1 is a key enzyme involved in maintaining sphingolipid rheostat in the brain. Deregulation of the sphingolipid metabolism may play a crucial role in the pathogenesis of Alzheimer’s disease (AD). Mitochondrial function and mitochondrial deacetylases, i.e. sirtuins (Sirt3,-4,-5), are also important for cell viability. In this study, we evaluated the interaction between Aβ_1–42, SphKs and Sirts in cell survival/death, and we examined several compounds to indicate possible target(s) for a strategy protecting against cytotoxicity of Aβ_1–42. PC12 cells were subjected to Aβ_1–42 oligomers and SphK inhibitor SKI II for 24–96 h. Our data indicated that Aβ_1–42 enhanced SphK1 expression and activity after 24 h, but down-regulated them after 96 h and had no effect on Sphk2. Aβ_1–42 and SKI II induced free radical formation, disturbed the balance between pro- and anti-apoptotic proteins and evoked cell death. Simultaneously, up-regulation of anti-oxidative enzymes catalase and superoxide dismutase 2 was observed. Moreover, the total protein level of glycogen synthase kinase-3β was decreased. Aβ_1–42 significantly increased the level of mitochondrial proteins: apoptosis-inducing factor AIF and Sirt3, -4, -5. By using several pharmacologically active compounds we showed that p53 protein plays a significant role at very early stages of Aβ_1–42 toxicity. However, during prolonged exposure to Aβ_1–42, the activation of caspases, MEK/ERK, and alterations in mitochondrial permeability transition pores were additional factors leading to cell death. Moreover, SphK product, sphingosine-1-phosphate (S1P), and Sirt activators and antioxidants, resveratrol and quercetin, significantly enhanced viability of cells subjected to Aβ_1–42. Our data indicated that p53 protein and inhibition of SphKs may be early key events responsible for cell death evoked by Aβ_1–42. We suggest that activation of S1P-dependent signalling and Sirts may offer a promising cytoprotective strategy.     

Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection

Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4⁺ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection.     

A RecA Protein Surface Required for Activation of DNA Polymerase V

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3''-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3''-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA''₂B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.