Structural and dynamical surface properties of phosphatidylethanolamine containing membranes

The hydration of solid dimyristoylphosphatidylethanolamine (DMPE) produces a negligible shift in the asymmetric stretching frequency of the phosphate groups in contrast to dimyristoylphosphatidylcholine (DMPC). This suggests that the hydration of DMPE is not a consequence of the disruption of the solid lattice of the phosphate groups as occurs in DMPC. The strong lateral interactions between NH₃ and PO₂⁻ groups present in the solid PEs remain when the lipids are fully hydrated and seem to be a limiting factor for the hydration of the phosphate group hindering the reorientation of the polar heads. The lower mobility is reflected in a higher energy to translocate the phosphoethanolamine (P-N) dipoles in an electrical field. This energy is decreased in the presence of increasing ratios of PCs of saturated chains in phosphoethanolamine monolayer. The association of PC and PE in the membrane affecting the reorientation of the P-N groups is dependent of the chain-chain interaction. The dipole potentials of PCs and PEs mixtures show different behaviors according to the saturation of the acyl chain. This was correlated with the area in monolayers and the hydration of the P-N groups. In spite of the low hydration, DMPE is still able to adsorb fully hydrated proteins, although in a lower rate than DMPC at the same surface pressure. This indicates that PE interfaces posses an excess of surface free energy to drive protein interaction. The relation of this free energy with the low water content is discussed.     

Multiplex genotyping and allele frequency estimation in pooled DNAs using non-gel capillary electrophoresis

We present a novel approach of single-nucleotide polymorphism (SNP) analysis in which allele-specific oligonucleotide hybridization is followed by non-gel capillary electrophoresis (ASOH-NGCE) in conjunction with laser-induced fluorescence (LIF). This allows rapid multiplex allelotyping and allele frequency estimation. This method, based on site separation of the hybridization duplexes, retains the simplicity and specificity of ASOH and the homogeneous feature of NGCE with poly(N,N-dimethylacrylamide) (PDMA) as a sieving medium. ASOH-NGCE can be applied to multiplex SNP loci genotyping with excellent separation of hybridization mixtures. Average relative standard deviations (RSDs) were low for within-day (1.10%) and between-day (2.41%) reproducibility. Moreover, the allele frequencies in pooled DNAs were accurately determined from peak areas and equilibrium dissociation constants. Our method was highly sensitive in detecting alleles with frequency as low as 1% and in distinguishing allele frequencies differing by 1% between pools. The average value of differences between real and estimated frequencies (accuracy) was only 0.004.     

The crystal structure of a hyperthermoactive exopolygalacturonase from Thermotoga maritima reveals a unique tetramer

The exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05 Å resolution. High structural homology around the active site allowed us to propose a model for substrate binding, explaining the exo-cleavage activity and specificity for non-methylated saturated galacturonate at the non-reducing end. Furthermore, the structure reveals unique features that contribute to the formation of stable tetramers in solution. Such an oligomerization has not been observed before for polygalacturonases.     

Acute kidney injury induced by protein-overload nephropathy down-regulates gene expression of hepatic cerebroside sulfotransferase in mice, resulting in reduction of liver and serum sulfatides

Sulfatides, possible antithrombotic factors belonging to sphingoglycolipids, are widely distributed in mammalian tissues and serum. We recently found that the level of serum sulfatides was significantly lower in hemodialysis patients than that in normal subjects, and that the serum level closely correlated to the incidence of cardiovascular disease. These findings suggest a relationship between the level of serum sulfatides and kidney function; however, the molecular mechanism underlying this relationship remains unclear. In the present study, the influence of kidney dysfunction on the metabolism of sulfatides was examined using an established murine model of acute kidney injury, protein-overload nephropathy in mice. Protein-overload treatment caused severe proximal tubular injuries within 4 days, and this treatment obviously decreased both serum and hepatic sulfatide levels. The sphingoid composition of serum sulfatides was very similar to that of hepatic ones at each time point, suggesting that the serum sulfatide level is dependent on the hepatic secretory ability of sulfatides. The treatment also decreased hepatic expression of cerebroside sulfotransferase (CST), a key enzyme in sulfatide metabolism, while it scarcely influenced the expression of the other sulfatide-metabolizing enzymes, including arylsulfatase A, ceramide galactosyltransferase, and galactosylceramidase. Pro-inflammatory responses were not detected in the liver of these mice; however, potential oxidative stress was increased. These results suggest that down-regulation of hepatic CST expression, probably affected by oxidative stress from kidney injury, causes reduction in liver and serum sulfatide levels. This novel mechanism, indicating the crosstalk between kidney injury and specific liver function, may prove useful for helping to understand the situation where human hemodialysis patients have low levels of serum sulfatides.     

Macrophages from alpha 7 nicotinic acetylcholine receptor knockout mice demonstrate increased cholesterol accumulation and decreased cellular paraoxonase expression: A possible link between the nervous system and atherosclerosis development

Objective

The parasympathetic nervous system regulates inflammation in peripheral tissues through a pathway termed the “cholinergic anti-inflammatory reflex” (CAIR). Mice deficient in the alpha 7 nicotinic acetylcholine receptor (α7^−/−) have an impaired CAIR due to decreased signaling through this pathway. The purpose of this study was to determine if the increased inflammation in α7^−/− mice is associated with enhanced serum and macrophage atherogenicity.

Methods

We measured serum markers of inflammation and oxidative stress, and macrophage atherogenicity in mouse peritoneal macrophages harvested from α7^−/− mice on the background of C57BL/6 mice, as well as on the background of the atherosclerotic Apolipoprotein E-deficient (ApoE^−/−) mice.

Results

α7-Deficiency had no significant effects on serum cholesterol, or on markers of serum oxidative stress (TBARS and paraoxonase1 activities). However, α7-deficiency significantly increased serum CRP and IL-6 (p < 0.05) levels in atherosclerotic mice, confirming an anti-inflammatory role for the α7 receptor. Macrophage cholesterol mass was increased by 25% in both normal and atherosclerotic mice in the absence of the α7 receptor (p < 0.05). This was accompanied by conditional increases in oxidized LDL uptake and in macrophage total peroxide levels. Furthermore, α7-deficiency reduced macrophage paraoxonase2 mRNA and activity by 50-100% in normal and atherosclerotic mice (p < 0.05 for each), indicating a reduction in macrophage anti-oxidant capacity in the α7^−/− mice.

Conclusion

The above results suggest an anti-atherogenic role for the macrophage α7nAchr, through a mechanism that involves attenuated macrophage oxidative stress and decreased uptake of oxidized LDL.     

Interspawning interval of wild female three-spined stickleback Gasterosteus aculeatus in Alaska

The interspawning interval, or spawning frequency, of wild three-spined stickleback, Gasterosteus aculeatus , was estimated using histological examination of postovulatory follicles (POF). Females in Alaskan lakes appeared to have as much as a 48 h delay between ovulation and ovoposition, yet the POF method could still be used to estimate the interspawning interval. In two Alaskan lakes the interspawning interval was estimated to range from 2·2 to 7·8 days among individual female G. aculeatus . These estimates were consistent with the range (2·5 to 5 days) of previous estimates among individual females from laboratory observations of spawning G. aculeatus , as well as anecdotal accounts of spawning intervals reported from wild populations in Canada (5–10 days). The interspawning interval of females increased during the course of the spawning season in Alaska, showing that the majority of female spawning activity occurred during the earliest portion of the approximate 6-week reproductive season. The increased interspawning interval appears to be related to a previously reported decrease in body condition in reproductive females during the breeding season. Thus, female G. aculeatus may be unable to sustain the initial rate of reproduction as energy stores that support the rapid growth of vitellogenic oocytes are depleted.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated co-transformation of multiple genes in upland cotton

Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes. However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different gene combinations as well as transformation frequency for different gene combinations. PCR analysis indicated that more than 80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. Southern blot analysis confirmed integration of target genes into the cotton genome, and the number of copies of the transgene(s) varied from one to four. Multiple transgene expression was confirmed by RT-PCR analysis in some transgenic lines. Further analysis of T₁ plants demonstrated that multiple transgenes were inherited and expressed in progenies. Fei-Fei Li and Shen-Jie Wu are joint first authors.     

The effects of allowance and frequency of allocation of deferred herbage, and grass silage feed value, when offered to ewes in mid-gestation on ewe and lamb performance and subsequent herbage yield

The effects of (i) herbage allowance, (ii) frequency of allocation and (iii) grass silage feed value on ewe and lamb performance were studied in mid-gestation ewes. Furthermore, the effects of (i) herbage allowance, (ii) frequency of allocation and (iii) grazing date and their interactions on subsequent herbage yield and feed value were also evaluated. Swards, which had a cut of silage removed on 6 September, received fertiliser nitrogen (34 kg/ha) for extended (deferred) grazing between 6 December and 1 February. Two grass silages differing in feed value were ensiled either precision chopped or in big bales from predominantly perennial ryegrass swards, respectively. In experiment 1, a completely randomised study involving 120 crossbred mid-gestation ewes (Belclare × Scottish Blackface) that had been mated in October was undertaken to evaluate the effects of extended grazed herbage allowance (1.0 and 1.8 kg dry matter (DM)/day), frequency of herbage allocation (daily and twice weekly) and grass silage feed value (low and medium) on ewe and subsequent lamb performance. The six diets were offered from days 63 to 120 of gestation. From day 120 of gestation to parturition all ewes were housed and offered the medium feed value silage ad libitum. All ewes received 19 kg concentrate prior to lambing. Increasing herbage allowance increased forage intake (P < 0.05), lamb birth weight (P < 0.01), weaning weight (P < 0.05) and growth rate from birth to weaning (P < 0.05), decreased herbage utilisation (P < 0.05) and tended to increase ewe condition score at lambing (P = 0.06). Frequency of herbage allocation or grass silage feed value did not alter (P > 0.05) ewe or subsequent lamb performance. In experiment 2, the effect of extended grazed herbage allowance (1.0 and 1.8 kg DM/ewe daily), frequency of allocation (daily and twice weekly) and grazing date (6 to 12 December, 27 December to 3 January and 17 to 23 January) on herbage yield at two harvest dates (27 April and 25 May) was examined in a split plot design study consisting of 72 plots. Delaying grazing date decreased herbage yield (P < 0.01) whilst delaying harvest date increased herbage yield (P < 0.05). Frequency of herbage allocation did not alter (P > 0.05) subsequent herbage yield. It is concluded that for ewes in mid-gestation 1.0 kg of low feed value silage DM had the same feed value, as determined by weaned lamb weight, as 1.3 kg herbage DM allowance. Each 1-day delay in grazing date reduced herbage DM yield by 54.2 kg/ha.     

The effects of different milking intervals and milking times per day in jennet milk production

In their first 150 days of lactation, nine Ragusana jennets were investigated at the 'Istituto Sperimentale Zootecnico per la Sicilia' (ISZS) in Palermo, to study the effects of different milking typologies on milk production. The jennets were kept in a paddock and were fed hay ad libitum and concentrate (3.5 kg jennet/day). From post-foaling day 21, every 3 weeks individual milk amounts were recorded, and individual milk samples were collected and analyzed for the main qualitative parameters. The compared theses were: two daily milking times with 6- and 3-h intervals; and two, three and eight daily milking times with 3-h interval. The jennets were manually milked. The foals were separated from the jennets at 0800 h, and after the last milking the foals were housed with the jennets. During the day with eight milkings, the milk yield from the jennets was fed to their respective foals, through bottles. The 6-h milking interval produced more milk (+19%) per session than the 3-h interval (P 0.01). The fat content per session, with the eight-time milking frequency (P 0.001), was greater than the others. For each milking typology, the lactation stage had a similar effect on almost all the considered variables. When observing the eight-milking times, the fat content (%) increased from 1100 to 0500 h (P 0.001). The milk yield and the fat percentage produced by two-, three- and eight-milking times were positively correlated.     

Fiber type homogeneity of the flight musculature in small birds

Studies of medium- and large-bodied avian species have suggested that variation in flight muscle composition is related to differences in flight behavior. For example, slow-twitch or tonic fibers are generally found only in the flight muscles of non-volant or soaring/gliding birds. However, we know comparatively little about fiber composition of the muscles of the smallest birds. Here we describe the fiber composition of muscles from the wings, shoulders, and legs of two small avian species, which also display very high wingbeat frequencies: Anna's hummingbirds (Calypte anna) and zebra finches (Taeniopygia guttata). All flight muscles examined in both species contained exclusively fast oxidative glycolytic (FOG) fibers. These unique results suggest that fast oxidative fibers are both necessary and sufficient for the full range of flight behaviors in these small-bodied birds. Like all other studied birds, the zebra finch gastrocnemius, a tarsometatarsal extensor, contained a mixture of FOG (27.1%), slow oxidative (SO, 12.7%), and fast glycolytic (FG, 60.2%) fibers. By contrast, the hummingbird gastrocnemius lacked FG fibers (85.5% FOG, 14.5% SO), which may reflect the reduced role of the hindlimb during take-off. We further hypothesize that thermogenic requirements constrain fiber type heterogeneity in these small endothermic vertebrates.