Shotgun proteomic analysis of cerebrospinal fluid using off-gel electrophoresis as the first-dimension separation

Shotgun proteomic analysis usually employs multidimensional separations with the first dimension most commonly being strong cation exchange (SCX) liquid chromatography (LC). SCX-LC is necessarily a serial process for preparation of multiple samples. Here, we apply a newly available tool, off-gel electrophoresis (OGE), for first-dimension separation of peptide mixtures from digests of cerebrospinal fluid (CSF), a complex and low total protein-containing sample. OGE first-dimension fractionation enabled identification of a total of 156 unique proteins compared to 115 identified in previous work using first-dimension SCX fractionation. OGE can be used to process multiple samples unattended with easy retrieval of the separated fractions. Thus, shotgun analysis using OGE as the first-dimension separation offers a significant advantage both in terms of sample throughput as well as increased numbers of identified proteins.     

Correlation between the content of intermediate chain-length fatty acids and copper in the milk of Fulani women

Intermediate chain-length fatty acids (C10-C14) in human milk triglycerides provide an easily absorbable fuel that provide a significant amount of the energy needed for growth during the first few months of life. The C10-C14 fatty acid and trace mineral content of human milk is variable. In this report we examined the relationship between the content of calcium, copper, magnesium, manganese, phosphorus, and zinc, and the weight percentage of C10-C14 fatty acids in milk from 33 Fulani women in northern Nigeria between 2 and 24 weeks post-gestation. The milk from these women contained proportions of C10-C14 fatty acids that were comparable to those reported for other populations around the world, as were the concentrations of Ca, Cu, Mg, Mn, Zn and P. Significant correlations were observed between the milk content of Cu and the wt% of C10 (P=0. 005, r=0.475), C12 (P=0.001, r=0.539), C14 (P=0.44, r=0.352) and the total intermediate chain-length fatty acids (P=0.008, r=0.450). No correlations were observed between these fatty acids and any of the other five minerals. We speculate that the relationship between Cu and fatty acids could be related to a requirement for Cu by an enzyme required for C10-C14 fatty acid biosynthesis (e.g. decanoyl deacylase) in mammary tissue, or to some unique Cu binding properties of the intermediate chain length fatty acids.     

T-B collaboration in the in vitro anti-Sm autoantibody response of MRL/Mp-lpr/lpr mice

Spontaneous production of autoantibodies to the Sm nuclear Ag is highly specific for SLE and the SLE-prone MRL mouse strains. Our previous studies have demonstrated that in vitro anti-Sm production in MRL/1pr mice requires the presence of T cells. In the present investigation, these T cells were found to express the L3T4+/Lyt-2- phenotype, unlike the aberrant L3T4-/Lyt-2-"double negative" 1pr T cells, and to utilize the L3T4 determinant in generating help for the anti-Sm response. The generation of anti-Sm did not require the presence of Sm-specific Th cells, as help could also be provided by T cells activated to an irrelevant Ag, or by nonspecific factors such as IL-2. There was no evidence for suppressor cell regulation of anti-Sm, even in animals negative for this specificity. These studies indicate that ongoing production of anti-Sm in MRL/1pr mice is dependent on the presence of T cells with a normal mature surface phenotype, and that these cells act in part through the elaboration of lymphokines. They further show that the anti-Sm status of individual MRL/1pr mice is not due to the action of suppressor cells. Because T cells appear to act primarily in a permissive fashion for the anti-Sm response, it is likely that events underlying the initial generation of Sm-specific B cell precursors are critical in determining whether an individual animal develops the Sm serologic specificity. Once these cells have arisen, clonal expansion of Sm-specific B cells may proceed in the presence of activated T cells or some of their products.     

Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives. From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Galalpha1-->6Glc. As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.      

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

The first range-wide assessment of Saddle-billed Stork Ephippiorhynchus senegalensis distribution

The Saddle-billed Stork Ephippiorhynchus senegalensis exemplifies a case in conservation research in which a species is assessed as Least Concern on the IUCN Red List and the resulting consideration of low conservation priority has precluded proper scientific study. As a first step in understanding this stork’s true status, we collated all available data to develop a distribution map and then investigated range-wide patterns of occurrence. The updated map greatly improves on past knowledge of the stork’s distribution and helps to identify regions where range contractions have occurred, particularly in Central Africa and parts of West Africa. We found that the stork’s distribution closely overlaps with protected areas and that there has been an overall increase in surface water (largely manmade water bodies)—a proxy for habitat—across the species’ extent of occurrence in recent decades. While this research represents a valuable contribution to our understanding of the Saddle-billed Stork, it also highlights the need for unbiased empirical data, especially from areas that are poorly surveyed, for developing a science-based conservation status assessment.     

Food habits of the world's grey wolves

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Three-Directional Evaluation of Mitral Flow in the Rat Heart by Phase-Contrast Cardiovascular Magnetic Resonance

Purpose

Determination of mitral flow is an important aspect in assessment of cardiac function. Traditionally, mitral flow is measured by Doppler echocardiography which suffers from several challenges, particularly related to the direction and the spatial inhomogeneity of flow. These challenges are especially prominent in rodents. The purpose of this study was to establish a cardiovascular magnetic resonance (CMR) protocol for evaluation of three-directional mitral flow in a rodent model of cardiac disease.

Materials and Methods

Three-directional mitral flow were evaluated by phase contrast CMR (PC-CMR) in rats with aortic banding (AB) (N = 7) and sham-operated controls (N = 7). Peak mitral flow and deceleration rate from PC-CMR was compared to conventional Doppler echocardiography. The accuracy of PC-CMR was investigated by comparison of spatiotemporally integrated mitral flow with left ventricular stroke volume assessed by cine CMR.

Results

PC-CMR portrayed the spatial distribution of mitral flow and flow direction in the atrioventricular plane throughout diastole. Both PC-CMR and echocardiography demonstrated increased peak mitral flow velocity and higher deceleration rate in AB compared to sham. Comparison with cine CMR revealed that PC-CMR measured mitral flow with excellent accuracy. Echocardiography presented significantly lower values of flow compared to PC-CMR.

Conclusions

For the first time, we show that PC-CMR offers accurate evaluation of three-directional mitral blood flow in rodents. The method successfully detects alterations in the mitral flow pattern in response to cardiac disease and provides novel insight into the characteristics of mitral flow.     

Ecogeochemistry potential in deep time biodiversity illustrated using a modern deep-water case study

The fossil record provides the only direct evidence of temporal trends in biodiversity over evolutionary timescales. Studies of biodiversity using the fossil record are, however, largely limited to discussions of taxonomic and/or morphological diversity. Behavioural and physiological traits that are likely to be under strong selection are largely obscured from the body fossil record. Similar problems exist in modern ecosystems where animals are difficult to access. In this review, we illustrate some of the common conceptual and methodological ground shared between those studying behavioural ecology in deep time and in inaccessible modern ecosystems. We discuss emerging ecogeochemical methods used to explore population connectivity and genetic drift, life-history traits and field metabolic rate and discuss some of the additional problems associated with applying these methods in deep time.     

Evolution of the mouse beta-globin genes: a recent gene conversion in the Hbbs haplotype

We have determined the complete nucleotide sequence of the two nonallelic adult beta-globin genes of the C57BL/10 mouse. These genes, designated beta s and beta t, show a sequence similarity of 99.6% over the region bordered by the translational start and stop codons. Both beta s and beta t encode functional polypeptide chains that are identical. A comparison of the C57BL/10 beta-globin haplotype, Hbbs, with that of the BALB/c mouse, Hbbd, suggests that the two haplotypes have distinct evolutionary histories. The two adult beta-globin genes of the Hbbd haplotype, beta dmaj and beta dmin, are 16% divergent at the nucleotide level and encode distinct polypeptides that are synthesized in differing amounts. Our analysis indicates that a gene correction mechanism has been operating on the Hbbs chromosome to keep beta s and beta t evolving in concert, whereas on the Hbbd chromosome, beta dmin has diverged considerably from beta dmaj. We suggest that gene conversion is responsible for the maintained similarity of the Hbbs genes. Furthermore, we attribute the divergence of the Hbbd genes in part to the absence of a region of simple-sequence DNA within the large intervening sequence of beta dmin. We propose that this region of DNA plays a role in facilitating gene conversion. The deletion of this area in beta dmin introduced a block of nonhomology between the beta dmaj-beta dmin gene pair and thus may have inhibited further gene correction within the Hbbd haplotype.