Structural studies on the O-specific side-chains of the cell-wall lipopolysaccharide from Escherichia coli O 75

The structure of the O-specific side-chains of the cell-wall lipopolysaccharide of Escherichia coli O 75 has been investigated, using methylation analysis and Smith degradation as the principal methods. The O-specific side-chain was found to be composed of a tetrasaccharide repeating-unit of the following structure:

     

The gamma protein specified by bacteriophage gamma. Structure and inhibitory activity for the recBC enzyme of Escherichia coli.

The protein encoded by the gam gene of bacteriophage lambda ("gamma protein") is a specific inhibitor of the recBC enzyme of Escherichia coli. The lambda protein has been purified approximately 2,000-fold, and its structure and inhibitory activity have been characterized. It appears to be composed of two identical subunits of 16,500 daltons, inhibits all of the catalytic activities of the recBC enzyme with apparently equal efficiency, but has no effect upon any other E. coli or lambda-DNase tested. Inhibition does not occur unless recBC enzyme is exposed to gamma protein prior to reaction of the enzyme with DNA. The inhibitory activity is independent of temperature, and no catalytic activity has been detected that might fulfill the inhibitory function. It appears instead that the inhibition involves a stoichiometric, rather than a catalytic interaction between gamma protein and the enzyme. Reaction kinetics for the recBC enzyme inhibited by gamma protein show no anomalous protein--only a depressed rate. Inhibition is not competitive and does not appear to affect the enzyme's affinity for DNA. The enzyme remains inhibited after it is separated from "excess" gamma protein by gel filtration or sedimentation in a glycerol gradient, and inhibited enzyme has a reduced electrophoretic mobility compared to that of uninhibited enzyme. Gamma Protein inhibits recBC enzyme which has been reconstituted from cell-free extracts by complementation in vitro, but at least one of the complementing factors present in extracts from recB- cells does not by itself form a complex with gamma protein. The mechanism of inhibition and the implications of these results from gamma replication and recombination are discussed.     

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.     

Sex-specific occurrence of androgen receptors in rat brain.

The metabolism and binding of [1, 2, 6, 7-3H] testosterone in male and female rat brain has been studied in an attempt to find an explanation for the relative androgen unresponsiveness characterizing the female hypothalamo-pituitary axis involved in regulation of hepatic steroid metabolism. The most significant sex differences in the pattern of [3H] testosterone metabolites recovered from several brain regions (including pituitary, pineal gland, and hypothalamus) after intraperitoneal administration of [3H] testosterone were the predominance of testosterone and androstenedione in male brain compared to the quantitative importance of 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, epitestosterone, and dihydroepitestosterone in female brain. One possible explanation for the androgen unresponsiveness of female rats is, therefore, the faster metabolism of testosterone to inactive compounds in female brain. Experiments both in vivo and in vitro showed the presence of high affinity, low capacity binding sites for [3H] testosterone in male pituitary, pineal gland, and hypothalamus (Kd values in the region of 1 X 10(-10) to 1 X 10(-9) M and number of binding sites 1.0 to 1.4 X 10(-14) mol per mg of protein). The steroid - macromolecular complexes generally had a pI of 5.1, were excluded from Sephadex G-200, were heat-labile, and were sensitive to protease. Competition experiments indicated the following order of ligand affinities: testosterone is greater than 5alpha-dihydrotestosterone and estradiol is greater than androstenedione is greater than corticosterone. No steroid-binding proteins of similar nature were found in pituitary, pineal gland, or hypothalamus from female rats. On the basis of these results it is suggested that the androgen unresponsiveness of female rats referred to above relates to the absence of receptor protein for androgens in female rat brain. In support of this hypothesis, 28-day-old female rats, which are known to be affected by androgens with regard to liver enzyme activities, were shown to contain receptor proteins for androgen in the brain. In conclusion, the relative androgen unresponsiveness of the female hypothalamo-pituitary axis is probably explained by the absence of receptor proteins for androgen in female hypothalamus and pituitary. The fast metabolism of testosterone in female rat brain also serves to decrease the availability of active androgen to potential receptor sites. It may be speculated that the presence of androgen receptors in male brain is the result of neonatal programming ("imprinting") by testicular androgen.     

Bovine Brain Myelin Glycerophosphocholine Choline Phosphodiesterase is an Alkaline Lysosphingomyelinase of the eNPP-Family, Regulated by Lysosomal Sorting

Glycerophosphocholine choline phosphodiesterase (GPC-Cpde) is a glycosylphosphatidylinositol (GPI)-anchored alkaline hydrolase that is expressed in the brain and kidney. In brain the hydrolase is synthesized by the oligodendrocytes and expressed on the myelin membrane. There are two forms of brain GPC-Cpde, a membrane-linked (mGPC-Cpde) and a soluble (sGPC-Cpde). Here we report the characterisation sGPC-Cpde from bovine brain. The amino acid sequence was identical to ectonucleotide pyrophosphatase/phosphodiesterase 6 (eNPP6) precursor, lacking the N-terminal signal peptide region and a C-terminal stretch, suggesting that the hydrolase was solubilised by C-terminal proteolysis, releasing the GPI-anchor. sGPC-Cpde existed as two isoforms, a homodimer joined by a disulfide bridge linking C414 from each monomer, and a monomer resulting from proteolysis N-terminally to this disulfide bond. The only internal disulfide bridge, linking C142 and C154, stabilises the choline-binding pocket. sGPC-Cpde was specific for lysosphingomyelin, displaying 1 to 2 orders of magnitude higher catalytic activity than towards GPC and lysophosphatidylcholine, suggesting that GPC-Cpde may function in the sphingomyelin signaling, rather than in the homeostasis of acylglycerophosphocholine metabolites. The truncated high mannose and bisected hybrid type glycans linked to N118 and N341 of sGPC-Cpde is a hallmark of glycans in lysosomal glycoproteins, subjected to GlcNAc-1-phosphorylation en route through Golgi. Thus, sGPC-Cpde may originate from the lysosomes, suggesting that lysosomal sorting contributes to the level of mGPC-Cpde on the myelin membrane.     

Sperm adaptation in relation to salinity in three goby species

In externally fertilizing species, the gametes of both males and females are exposed to the influences of the environment into which they are released. Sperm are sensitive to abiotic factors such as salinity, but they are also affected by biotic factors such as sperm competition. In this study, the authors compared the performance of sperm of three goby species, the painted goby, Pomatoschistus pictus, the two-spotted goby, Pomatoschistus flavescens, and the sand goby, Pomatoschistus minutus. These species differ in their distributions, with painted goby having the narrowest salinity range and sand goby the widest. Moreover, data from paternity show that the two-spotted goby experiences the least sperm competition, whereas in the sand goby sperm competition is ubiquitous. The authors took sperm samples from dissected males and exposed them to high salinity water (31 PSU) representing the North Sea and low salinity water (6 PSU) representing the brackish Baltic Sea Proper. They then used computer-assisted sperm analysis to measure the proportion of motile sperm and sperm swimming speed 10 min and 20 h after sperm activation. The authors found that sperm performance depended on salinity, but there seemed to be no relationship to the species' geographical distribution in relation to salinity range. The species differed in the proportion of motile sperm, but there was no significant decrease in sperm motility during 20 h. The sand goby was the only species with motile sperm after 72 h.     

ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq

Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.     

Courtship Sounds Advertise Species Identity and Male Quality in Sympatric Pomatoschistus spp. Gobies

Acoustic signals can encode crucial information about species identity and individual quality. We recorded and compared male courtship drum sounds of the sand goby Pomatoschistus minutus and the painted goby P. pictus and examined if they can function in species recognition within sympatric populations. We also examined which acoustic features are related to male quality and the factors that affect female courtship in the sand goby, to determine whether vocalisations potentially play a role in mate assessment. Drums produced by the painted goby showed significantly higher dominant frequencies, higher sound pulse repetition rates and longer intervals between sounds than those of the sand goby. In the sand goby, male quality was predicted by visual and acoustic courtship signals. Regression analyses showed that sound amplitude was a good predictor of male length, whereas the duration of nest behaviour and active calling rate (i.e. excluding silent periods) were good predictors of male condition factor and fat reserves respectively. In addition, the level of female courtship was predicted by male nest behaviour. The results suggest that the frequency and temporal patterns of sounds can encode species identity, whereas sound amplitude and calling activity reflects male size and fat reserves. Visual courtship duration (nest-related behaviour) also seems relevant to mate choice, since it reflects male condition and is related to female courtship. Our work suggests that acoustic communication can contribute to mate choice in the sand goby group, and invites further study.     

Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen''s (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)²; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction² and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations³. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.     

Long-Term Effect of Bariatric Surgery on Liver Enzymes in the Swedish Obese Subjects (SOS) Study

Background and Aim

Obesity is associated with elevated serum transaminase levels and non-alcoholic fatty liver disease and weight loss is a recommended therapeutic strategy. Bariatric surgery is effective in obtaining and maintaining weight loss. Aim of the present study was to examine the long-term effects of bariatric surgery on transaminase levels in obese individuals.

Methods

The Swedish Obese Subjects (SOS) study is a prospective controlled intervention study designed to compare the long-term effects of bariatric surgery and usual care in obese subjects. A total of 3,570 obese participants with no excess of alcohol consumption at baseline (1,795 and 1,775 in the control and surgery group, respectively) were included in the analyses. Changes in transaminase levels during follow-up were compared in the surgery and control groups.

Results

Compared to usual care, bariatric surgery was associated with lower serum ALT and AST levels at 2- and 10- year follow up. The reduction in ALT levels was proportional to the degree of weight loss. Both the incidence of and the remission from high transaminase levels were more favorable in the surgery group compared to the control group. Similarly, the prevalence of ALT/AST ratio <1 was lower in the surgery compared to the control group at both 2- and 10-year follow up.

Conclusions

Bariatric surgery results in a sustained reduction in transaminase levels and a long-term benefit in obese individuals.