Developing functional magnetic resonance imaging techniques for alert macaque monkeys

Functional magnetic resonance imaging (fMRI) has developed rapidly into a major non-invasive tool for studying the human brain. However, due to a variety of technical difficulties, it has yet to be widely adopted for use in alert, trained non-human primates. Our laboratory has been developing techniques for such fMRI studies. As background, we first consider basic principles of fMRI imaging, experimental design, and post-processing. We discuss appropriate MRI system hardware and components for conducting fMRI studies in alert macaques, and the animal preparation and behavior necessary for optimal experiments. Finally, we consider alternative fMRI techniques using exogenous contrast agents, arterial spin labeling, and more direct measures of neural activation.     

The effects of kefir,koumiss, yogurt and commercial probiotic formulations on PPARα and PPAR-β/δ expressions in mouse kidney

Commercial probiotic capsules that contain probiotic bacteria, kefir, koumiss and yogurt contain beneficial microorganisms that affect cholesterol levels and immune response, and are used for treatment of some diseases. We investigated using immunohistochemistry the effects of kefir, koumiss, yogurt and a commercial probiotic formulation on the expression levels of peroxisome proliferator-activated receptor-α (PPARα) and peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ), which are members of the nuclear steroid hormone receptor superfamily in mouse kidney. Mice were assigned to five groups: group 1, commercial probiotic capsules; group 2, kefir; group 3, koumiss; group 4, yogurt; group 5, control. After oral administration for 15 days, body weights were recorded and kidney tissue samples were obtained. Hematoxylin & eosin staining and the streptavidin-biotin peroxidase complex (ABC) method were applied to tissue sections to examine histology and to determine the localization of PPARα and PPAR-β/δ in the kidneys. We found that the weights of the mice in the kefir, koumiss, yogurt and commercial probiotic capsules groups were increased compared to controls. No differences in kidney histology were observed in any of the experimental groups. Kefir, koumiss, yogurt and the commercial probiotic preparation increased PPARα and PPAR-β/δ expressions.     

The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.     

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.     

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: II. Distribution of radioactive peptide hormones and hormone precursors in subcellular fractions after pulse and pulse- chase incubation of islet tissue

Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.     

Ectomycorrhizal fungal succession in mixed temperate forests

Ectomycorrhizal (ECM) fungal communities of Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyrifera) were studied along a chronosequence of forest development after stand-replacing disturbance. Previous studies of ECM succession did not use molecular techniques for fungal identification or lacked replication, and none examined different host species. Four age classes of mixed forests were sampled: 5-, 26-, 65-, and 100-yr-old, including wildfire-origin stands from all four classes and stands of clearcut origin from the youngest two classes. Morphotyping and DNA sequences were used to identify fungi on ECM root tips. ECM fungal diversities were lower in 5-yr-old than in older stands on Douglas-fir, but were similar among age classes on paper birch. Host-specific fungi dominated in 5-yr-old stands, but host generalists were dominant in the oldest two age classes. ECM fungal community compositions were similar in 65- and 100-yr-old stands but differed among all other pairs of age classes. Within the age range studied, site-level ECM fungal diversity reached a plateau by the 26-yr-old age class, while community composition stabilized by the 65-yr-old class. Simple categories such as 'early stage', 'multi stage', and 'late stage' were insufficient to describe fungal species' successional patterns. Rather, ECM fungal succession may be best described in the context of stand development.     

Sperm surface galactosyltransferase activities during in vitro capacitation

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as “decapacitation factors” when added back to in vitro fertilization assays. These glycoside “decapacitation factors” inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces “decapacitation factio” competition. On the other hand “conventional” low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of “decapacitation factos” is a necessary prerequisite for sperm binding to the zona pellucida.     

Influence of soil nutrients on ectomycorrhizal communities in a chronosequence of mixed temperate forests

Many factors associated with forests are collectively responsible for controlling ectomycorrhizal (ECM) fungal community structure, including plant species composition, forest structure, stand age, and soil nutrients. The objective of this study was to examine relationships among ECM fungal community measures, local soil nutrients, and stand age along a chronosequence of mixed forest stands that were similar in vegetation composition and site quality. Six combinations of age class (5-, 26-, 65-, and 100-year-old) and stand initiation type (wildfire and clearcut) were replicated on four sites, each representing critical seral stages of stand development in Interior Cedar-Hemlock (ICH) forests of southern British Columbia. We found significant relationships between ECM fungal diversity and both available and organic P; available P was also positively correlated with the abundance of two ECM taxa (Rhizopogon vinicolor group and Cenoccocum geophilum). By contrast, ECM fungal diversity varied unpredictably with total and mineralizable N or C to N ratio. We also found that soil C, N, available P, and forest floor depth did not exhibit strong patterns across stand ages. Overall, ECM fungal community structure was more strongly influenced by stand age than specific soil nutrients, but better correlations with soil nutrients may occur at broader spatial scales covering a wider range of site qualities.