Assembly of an RNA-protein complex. Binding of NusB and NusE (S10) proteins to boxA RNA nucleates the formation of the antitermination complex involved in controlling rRNA transcription in Escherichia coli

Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K(d) values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K(d) values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available single-stranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.     

Seaweed processing using industrial single-mode cavity microwave heating: a preliminary investigation

A single-cavity microwave heating system has been designed and fabricated for microwave-assisted extraction of carrageenans from seaweed. The system comprises a single mode (TE101) waveguide fitted with power and temperature controls, together with a continuous-flow-recycle reactor operating at atmospheric pressure. The system has been tested by extraction of E. cottonii and E. spinosum in aqueous organic solvents. Even without purification, the extraction products were found to have virtually identical FTIR and 13C and 1H NMR spectra to the reference samples of kappa- and iota-carrageenan, respectively. The principal advantages of the microwave system are substantial reduction of extraction time and low consumption of organic solvents.     

Nereis diversicolor effect on the stability of cohesive intertidal sediments

The effect of the polychaete Nereis diversicolor on the stability of natural cohesive sediments was investigated in the laboratory. Three densities (450, 600 and 1200 ind m⁻²) of N. diversicolor were used. Sediment shear strength was measured using a cone penetrometer. Sediment erodability was assessed using an annular flume (current velocities from 5 to 55 cm s⁻¹) in which flow velocity was increased incrementally, and water sampled to quantify suspended material in order to derive critical erosion velocity and erosion rates. At low current velocities ( <25 cm s⁻¹), we found N. diversicolor to have a stabilising effect, reflected by an increase of up to 20% in the critical erosion velocity. This is related to an enhancement of ~50% in shear strength, due probably to gallery building activities, responsible for the promotion of lateral compaction, an increase in the area of the sediment–water interface, and enhanced microphytobenthos production. Once the sediment began to erode, the stabilising effect of N. diversicolor reverses, leading to an increase of up to 40% in eroded matter due to compaction, which resulted in the erosion of larger aggregates. The balance between the effect of N. diversicolor on herbivory and microphytobenthos production due to the presence of galleries is discussed. Our results indicate that neither chlorophyll a, nor shear strength nor critical erosion velocity are good indicators of erodability. This underlines the need to include biogeochemical processes in any realistic sediment transport model.     

Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of Legionellae in biofilms

Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.     

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of ∼7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 μg porcine aprotinin inhibited 6 μg trypsin whereas 1 μg commercial soybean inhibitor inhibited only 1 μg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.     

Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.     

Volatiles released from foliar extract of host plant enhance landing rates of gravid Polygonia c‐album females,but do not stimulate oviposition

The role of olfactory cues for host search is much less investigated in day‐active butterflies than in their relatives, the nocturnal moths. The goal of this study was to investigate whether host‐plant volatiles from foliar extracts of hop, Humulus lupulus L. (Cannabaceae), evoke electroantennographic (EAG) responses, increase landing rates, and stimulate egg‐laying behavior of gravid Polygonia c‐album L. (Lepidoptera: Nymphalidae) females. Eighty‐nine volatile compounds were detected in a non‐concentrated methanol extract of hop by gas chromatography–mass spectrometry, 11 of which elicited an EAG response. Concentration of the crude extract significantly reduced landing rates on artificial leaves treated with the sample due to loss of volatile compounds, but after landing the oviposition response of gravid females was not affected. A mixture of eight commercially available EAG‐active volatiles increased the landing rate of gravid females to their source but did not act as oviposition stimulants. Dividing the volatile compounds into two groups – consisting of (1) hexanal, (E)‐2‐hexenal, octanal, nonanal, and decanal, and (2) sulcatone, humulene, and benzyl alcohol – obliterated effectiveness, revealing synergism between compounds. Although volatiles did not stimulate oviposition, they significantly contributed to the distribution of eggs by increasing the landing rates on treated artificial leaves.     

Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain

Neurofibrillary tangles (NFT) and β-amyloid plaques are the neurological hallmarks of both Alzheimer''s disease and an unusual paralytic illness suffered by Chamorro villagers on the Pacific island of Guam. Many Chamorros with the disease suffer dementia, and in some villages one-quarter of the adults perished from the disease. Like Alzheimer''s, the causal factors of Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) are poorly understood. In replicated experiments, we found that chronic dietary exposure to a cyanobacterial toxin present in the traditional Chamorro diet, β-N-methylamino-l-alanine (BMAA), triggers the formation of both NFT and β-amyloid deposits similar in structure and density to those found in brain tissues of Chamorros who died with ALS/PDC. Vervets (Chlorocebus sabaeus) fed for 140 days with BMAA-dosed fruit developed NFT and sparse β-amyloid deposits in the brain. Co-administration of the dietary amino acid l-serine with l-BMAA significantly reduced the density of NFT. These findings indicate that while chronic exposure to the environmental toxin BMAA can trigger neurodegeneration in vulnerable individuals, increasing the amount of l-serine in the diet can reduce the risk.     

Quantitative Timed-Up-and-Go Parameters in Relation to Cognitive Parameters and Health-Related Quality of Life in Mild-to-Moderate Parkinson's Disease

IntroductionThe instrumented-Timed-Up-and-Go test (iTUG) provides detailed information about the following movement patterns: sit-to-walk (siwa), straight walking, turning and walk-to-sit (wasi). We were interested in the relative contributions of respective iTUG sub-phases to specific clinical deficits most relevant for daily life in Parkinson’s disease (PD). More specifically, we investigated which condition–fast speed (FS) or convenient speed (CS)–differentiates best between mild- to moderate-stage PD patients and controls, which parameters of the iTUG sub-phases are significantly different between PD patients and controls, and how the iTUG parameters associate with cognitive parameters (with particular focus on cognitive flexibility and working memory) and Health-Related-Quality of Life (HRQoL).MethodsTwenty-eight PD participants (65.1±7.1 years, H&Y stage 1–3, medication OFF state) and 20 controls (66.1±7.5 years) performed an iTUG (DynaPort^®, McRoberts BV, The Netherlands) under CS and FS conditions. The PD Questionnaire 39 (PDQ-39) was employed to assess HRQoL. General cognitive and executive functions were assessed using the Montreal Cognitive Assessment and the Trail Making Test.ResultsThe total iTUG duration and sub-phases durations under FS condition differentiated PD patients slightly better from controls, compared to the CS condition. The following sub-phases were responsible for the observed longer total duration PD patients needed to perform the iTUG: siwa, turn and wasi. None of the iTUG parameters correlated relevantly with general cognitive function. Turning duration and wasi maximum flexion velocity correlated strongest with executive function. Walking back duration correlated strongest with HRQoL.DiscussionThis study confirms that mild- to moderate-stage PD patients need more time to perform the iTUG than controls, and adds the following aspects to current literature: FS may be more powerful than CS to delineate subtle movement deficits in mild- to moderate-stage PD patients; correlation levels of intra-individual siwa and wasi parameters may be interesting surrogate markers for the level of automaticity of performed movements; and sub-phases and kinematic parameters of the iTUG may have the potential to reflect executive functioning and HRQoL aspects of PD patients.