Molecular models and structural comparisons of native and mutant class I filamentous bacteriophages Ff (fd, f1, M13), If1 and IKe

The filamentous bacteriophages are flexible rods about 1 to 2 microns long and 6 nm in diameter, with a helical shell of protein subunits surrounding a DNA core. The approximately 50-residue coat protein subunit is largely alpha-helix and the axis of the alpha-helix makes a small angle with the axis of the virion. The protein shell can be considered in three sections: the outer surface, occupied by the N-terminal region of the subunit, rich in acidic residues that interact with the surrounding solvent and give the virion a low isoelectric point; the interior of the shell, including a 19-residue stretch of apolar side-chains, where protein subunits interact mainly with each other; and the inner surface, occupied by the C-terminal region of the subunit, rich in basic residues that interact with the DNA core. The fact that virtually all protein side-chain interactions are between different subunits in the coat protein array, rather than within subunits, makes this a useful model system for studies of interactions between alpha-helix subunits in a macromolecular assembly. We describe molecular models of the class I filamentous bacteriophages. This class includes strains fd, f1, M13 (these 3 very similar strains are members of the Ff group), If1 and IKe. Our model of fd has been refined to fit quantitative X-ray fibre diffraction data to 30 A resolution in the meridional direction and 7 A resolution in the equatorial direction. A simulated 3.3 A resolution diffraction pattern from this model has the same general distribution of intensity as the experimental diffraction pattern. The observed diffraction data at 7 A resolution are fitted much better by the calculated diffraction pattern of our molecular model than by that of a model in which the alpha-helix subunit is represented by a rod of uniform density. The fact that our fd model explains the fd diffraction data is only part of our structure analysis. The atomic details of the model are supported by non-diffraction data, in part previously published and in part newly reported here. These data include information about permitted or forbidden side-chain replacements, about the effect of chemical modification, and about spectroscopic experiments.(ABSTRACT TRUNCATED AT 400 WORDS

Determination of picomolar dissolved free amino acids along a South Atlantic transect using reversed-phase high-performance liquid chromatography

Dissolved free amino acids (DFAA) in seawater are a form of nitrogen (N) available for marine microbes. In oligotrophic environments where N-containing nutrients are the limiting factor for microbial growth, N nutrition from DFAA could be crucial, but as yet it is poorly resolved. Measurements of individual DFAA are challenging as concentrations are typically in the low nmol L− 1 range. Here we report modifications to methodology using o-phthaldialdehyde (OPA) derivatization and reversed phase high performance liquid chromatography (HPLC) that provide a 30-fold improvement in sensitivity enabling the detection of 15 amino acids in seawater with a limit of detection as low as 10 pmol L− 1 with accuracy and precision of better than 10%. This analytical methodology is now suitable for the challenging quantitation of DFAA in oligotrophic seawaters. The method was successfully applied to a suite of seawater samples collected on a cruise crossing the South Atlantic Ocean, where concentrations of DFAAs were generally low (sub nmol L− 1), revealing basin-scale features in the oceanographic distributions of DFAA. This unique dataset implies that DFAAs are an important component of the N cycle in both near-coastal and open oceans. Further calculations suggest that the proportions of organic N originating from DFAA sources were significant, contributing between 0.2 and 200% that of NH4+ and up to 77% that of total inorganic nitrogen in the upper 400 m in some regions of the transect

The breast feeding mother and xenon anaesthesia: four case reports. Breast feeding and xenon anaesthesia

<p>Abstract</p> <p>Background</p> <p>Four nursing mothers consented to anaesthesia for urgent surgery only on condition that their ability to breast feed would not be impaired.</p> <p>Methods</p> <p>Following induction of general anaesthesia with propofol and remifentanil, 65-69% xenon supplemented with remifentanil was used as an inhalational anaesthetic for maintenance.</p> <p>Results</p> <p>After finishing surgery the women could be extubated between 2:52 and 7:22 minutes. The women were fully alert just minutes after extubation and spent about 45 minutes in the recovery room before discharge to a regular ward. They resumed regular breast feeding some time later. The propofol concentration in the blood was measured after 0, 30, 90, and 300 minutes and in the milk after 90 and 300 minutes. Just 90 minutes after extubation, the concentration of propofol in the milk was limited (> 3 mg/l) so that pharmacological effects on the babies were excluded after oral intake. Also, no traces of xenon gas were found in the maternal milk at any time. After propofol induction and maintenance of anaesthesia with xenon in combination with a water-soluble short-acting drug like remifentanil, the concentration of propofol in maternal milk is low (> 3 mg/l 90 min after anesthesia) and harmless after oral intake.</p> <p>Conclusions</p> <p>These results, as well as the rapid elimination and absence of metabolism of xenon, are of great interest to nursing mothers. General anaesthesia with propofol for induction only, combined with remifentanil and xenon for maintenance, has not yet been described in breast feeding mothers.</p

Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT3 receptor antagonists

PURPOSE: The ferret cisplatin emesis model has been used for ~30 years and enabled identification of clinically used anti-emetics. We provide an objective assessment of this model including efficacy of 5-HT(3) receptor antagonists to assess its translational validity. METHODS: A systematic review identified available evidence and was used to perform meta-analyses. RESULTS: Of 182 potentially relevant publications, 115 reported cisplatin-induced emesis in ferrets and 68 were included in the analysis. The majority (n = 53) used a 10 mg kg(−1) dose to induce acute emesis, which peaked after 2 h. More recent studies (n = 11) also used 5 mg kg(−1), which induced a biphasic response peaking at 12 h and 48 h. Overall, 5-HT(3) receptor antagonists reduced cisplatin (5 mg kg(−1)) emesis by 68% (45–91%) during the acute phase (day 1) and by 67% (48–86%) and 53% (38–68%, all P < 0.001), during the delayed phase (days 2, 3). In an analysis focused on the acute phase, the efficacy of ondansetron was dependent on the dosage and observation period but not on the dose of cisplatin. CONCLUSION: Our analysis enabled novel findings to be extracted from the literature including factors which may impact on the applicability of preclinical results to humans. It reveals that the efficacy of ondansetron is similar against low and high doses of cisplatin. Additionally, we showed that 5-HT(3) receptor antagonists have a similar efficacy during acute and delayed emesis, which provides a novel insight into the pharmacology of delayed emesis in the ferret

Management Impacts on Forest Floor and Soil Organic Carbon in Northern Temperate Forests of the US

<p>Abstract</p> <p>Background</p> <p>The role of forests in the global carbon cycle has been the subject of a great deal of research recently, but the impact of management practices on forest soil dynamics at the stand level has received less attention. This study used six forest management experimental sites in five northern states of the US to investigate the effects of silvicultural treatments (light thinning, heavy thinning, and clearcutting) on forest floor and soil carbon pools.</p> <p>Results</p> <p>No overall trend was found between forest floor carbon stocks in stands subjected to partial or complete harvest treatments. A few sites had larger stocks in control plots, although estimates were often highly variable. Forest floor carbon pools did show a trend of increasing values from southern to northern sites. Surface soil (0-5 cm) organic carbon content and concentration were similar between treated and untreated plots. Overall soil carbon (0-20 cm) pool size was not significantly different from control values in sites treated with partial or complete harvests. No geographic trends were evident for any of the soil properties examined.</p> <p>Conclusions</p> <p>Results indicate that it is unlikely that mineral soil carbon stocks are adversely affected by typical management practices as applied in northern hardwood forests in the US; however, the findings suggest that the forest floor carbon pool may be susceptible to loss.</p

Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli

BACKGROUND: Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library. RESULTS: Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. CONCLUSION: Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes

The Early Apoptotic DNA Fragmentation Targets a Small Number of Specific Open Chromatin Regions

We report here that early apoptotic DNA fragmentation, as obtained by using an entirely new approach, is the result of an attack at a small number of specific open chromatin regions of interphase nuclei. This was demonstrated as follows: (i) chicken liver was excised and kept in sterile tubes for 1 to 3 hours at 37°C; (ii) this induced apoptosis (possibly because of oxygen deprivation), as shown by the electrophoretic nucleosomal ladder produced by DNA preparations; (iii) low molecular-weight DNA fragments (∼200 bp) were cloned, sequenced, and shown to derive predominantly from genes and surrounding 100 kb regions; (iv) a few hundred cuts were produced, very often involving the same chromosomal sites; (v) at comparable DNA degradation levels, micrococcal nuclease (MNase) also showed a general preference for genes and surrounding regions, but MNase cuts were located at sites that were quite distinct from, and less specific than, those cut by apoptosis. In conclusion, the approach presented here, which is the mildest and least intrusive approach, identifies a preferred accessibility landscape in interphase chromatin

Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1

Peer reviewedPublisher PD

Mild hypothermia reduces cardiac post-ischemic reactive hyperemia

BACKGROUND: In experimentally induced myocardial infarction, mild hypothermia (33–35°C) is beneficial if applied prior to ischemia or reperfusion. Hypothermia, when applied after reperfusion seems to confer little or no benefit. The mechanism by which hypothermia exerts its cell-protective effect during cardiac ischemia remains unclear. It has been hypothesized that hypothermia reduces the reperfusion damage; the additional damage incurred upon the myocardium during reperfusion. Reperfusion results in a massive increase in blood flow, reactive hyperemia, which may contribute to reperfusion damage. We postulated that hypothermia could attenuate the post-ischemic reactive hyperemia. METHODS: Sixteen 25–30 kg pigs, in a closed chest model, were anesthetized and temperature was established in all pigs at 37°C using an intravascular cooling catheter. The 16 pigs were then randomized to hypothermia (34°C) or control (37°C). The left main coronary artery was then catheterized with a PCI guiding catheter. A Doppler flow wire was placed in the mid part of the LAD and a PCI balloon was then positioned proximal to the Doppler wire but distal to the first diagonal branch. The LAD was then occluded for ten minutes in all pigs. Coronary blood flow was measured before, during and after ischemia/reperfusion. RESULTS: The peak flow seen during post-ischemic reactive hyperemia (during the first minutes of reperfusion) was significantly reduced by 43 % (p < 0.01) in hypothermic pigs compared to controls. CONCLUSION: Mild hypothermia significantly reduces post-ischemic hyperemia in a closed chest pig model. The reduction of reactive hyperemia during reperfusion may have an impact on cardiac reperfusion injury