CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.     

Ultrastructural immunocytochemical localization of neurophysin and vasopressin in the median eminence and posterior pituitary of the guinea pig

Summary With the use of tissue prepared by freeze-substitution and the unlabelled antibody enzyme technique, neurophysin and vasopressin were localized at the ultrastructural level in the posterior pituitary and median eminence of the guinea pig. In the posterior pituitary neurophysin was found in the large neurosecretory granules (1300–1500 Å) of axons, Herring bodies, and nerve terminals. In some of these axons immunoreactive neurophysin was found outside of granules in the axoplasm. By light microscopy neurophysin was found in both the zona interna and zona externa of the median eminence; this was confirmed by electron microscopy. In the zona interna as in the posterior pituitary, neurophysin was localized both inside and outside the large neurosecretory granules. In the zona externa, immunoreactive deposit was primarily located in granules with a diameter of 900–1100 Å in nerve terminals abutting on the primary portal plexus. The distribution of vasopressin paralleled that of neurophysin except that the hormone was rarely extragranular. These results demonstrate for the first time that both neurophysin and vasopressin are present in granules of axons that are in contact with the hypophysial portal vasculature.The authors wish to thank Dr. Alan Robinson for the gifts of antiserum to bovine neurophysin I and for purified bovine neurophysin I; Dr. Ludwig Sternberger for the peroxidase-anti-peroxidase complex; and Dr. Robert Utiger for antiserum to lysine vasopressinSupported in part by U.S. Public Health Service grant RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center publication No. 14-017.Recipient of NIH, NINDS Teacher-Investigator Award NS-1108.     

Tubulin dimer dissociation and proteolytic accessibility

The alpha and beta subunits of the tubulin dimer each possess a distal C-terminal subtilisin cleavage site which, when cleaved, releases an acidic, small peptide. In addition, each possesses an internal site, cleaved by trypsin in alpha and chymotrypsin in beta, which connects the amino and carboxyl structural domains. A model of the dimer is presented which suggests that the beta C-terminal subtilisin site may be more accessible in the monomer than in the dimer. Kinetics of cleavage at this site on the dimer yield straight-line plots of log (undigested fraction) versus time, from which pseudo-first-order rate constants are obtained. Temperature effects on the rate constant are due to changes in the activity of subtilisin, not to temperature-induced unfolding around this site. The rate constant is proportional to the subtilisin/tubulin ratio, whether this is varied by changing the concentration of subtilisin or of tubulin. However, if the rate constant increases due to decreasing tubulin concentration, the extrapolated zero time intercept decreases. The decrease in zero time intercept is interpreted as being due to the appearance of a rapidly digested fraction upon dilution of tubulin. The increase observed in this fast fraction with dilution of tubulin is fully reversible upon reconcentration. It is suggested that this fast fraction represents monomeric beta-tubulin and the concentration dependence of this fast fraction indicates a dissociation constant of about 1.5 X 10(-7) M.     

Effects of diet,habitat, and phylogeny on the fecal microbiome of wild African savanna (Loxodonta africana) and forest elephants (L. cyclotis)

The gut microbiome, or the community of microorganisms inhabiting the digestive tract, is often unique to its symbiont and, in many animal taxa, is highly influenced by host phylogeny and diet. In this study, we characterized the gut microbiome of the African savanna elephant (Loxodonta africana) and the African forest elephant (Loxodonta cyclotis), sister taxa separated by 2.6–5.6 million years of independent evolution. We examined the effect of host phylogeny on microbiome composition. Additionally, we examined the influence of habitat types (forest versus savanna) and diet types (crop‐raiding versus noncrop‐raiding) on the microbiome within L. africana. We found 58 bacterial orders, representing 16 phyla, across all African elephant samples. The most common phyla were Firmicutes, Proteobacteria, and Bacteroidetes. The microbiome of L. africana was dominated by Firmicutes, similar to other hindgut fermenters, while the microbiome of L. cyclotis was dominated by Proteobacteria, similar to more frugivorous species. Alpha diversity did not differ across species, habitat type, or diet, but beta diversity indicated that microbial communities differed significantly among species, diet types, and habitat types. Based on predicted KEGG metabolic pathways, we also found significant differences between species, but not habitat or diet, in amino acid metabolism, energy metabolism, and metabolism of terpenoids and polyketides. Understanding the digestive capabilities of these elephant species could aid in their captive management and ultimately their conservation.     

Environmental gradients drive physiological variation in Hawaiian corals

Coral Reefs - To evaluate potential coral adaptive mechanisms, we investigated physiological traits (biomass, lipid, protein, chlorophyll, and isotopic proxies for trophic strategy) in eight...     

Comparative analysis of 13C chemical shifts of β-sheet amyloid proteins and outer membrane proteins

Cross-β amyloid fibrils and membrane-bound β-barrels are two important classes of β-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the ¹³C chemical shifts of 17 amyloid proteins and 7 β-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 β-sheet residues in amyloid fibrils and 521 β-sheet residues in β-barrel membrane proteins. The ¹³C chemical shifts are shown in 2D ¹³C–¹³C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in β-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and β-barrels and compare the sidechain rotameric populations. The ¹³C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in β-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in β-barrels. These trends can be explained by steric zipper interactions between β-sheet planes in cross-β fibrils, and by the interactions of β-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and β-barrels based principally on NMR chemical shifts.

     

Rejoinder to Discussions on “Approval policies for modifications to machine learning‐based software as a medical device: A study of bio‐creep”

We thank the discussants for sharing their unique perspectives on the problem of designing automatic algorithm change protocols (aACPs) for machine learning‐based software as a medical device. Both Pennello et al. and Rose highlighted a number of challenges that arise in real‐world settings, and we whole‐heartedly agree that substantial extensions of our work are needed to understand if and how aACPs can be safely deployed in practice. Our work demonstrated that aACPs that appear to be harmless may allow for biocreep, even when the data distribution is assumed to be representative and stationary over time. While we investigated two solutions that protect against this specific issue, many more statistical and practical challenges remain and we look forward to future research on this topic.