Species delimitation of the liquorice tribe (Leguminosae: Glycyrrhizeae) based on phylogenomic and machine learning analyses

The liquorice tribe Glycyrrhizeae is a leguminous herbaceous group of plants comprised of the genera Glycyrrhiza and Glycyrrhizopsis. Some Glycyrrhiza taxa contain glycyrrhizin, a pharmacologically significant sweet substance that also has applications in crafting industrial materials. Here, we utilized an expanded taxon sampling of Glycyrrhizeae to reconstruct the phylogenetic relationships in the tribe based on genome skimming data, including whole chloroplast genomes, nuclear ribosomal DNA, and low-copy nuclear DNA. We also launched machine learning analysis (MLA) for one species pair with controversial taxonomic boundary. The integrated results indicated Glycyrrhizopsis should be split from Glycyrrhiza, while the former genus Meristotropis should be treated as part of Glycyrrhiza. Glycyrrhizopsis includes two species, Glycyrrhizopsis asymmetrica and Glycyrrhizopsis flavescens, and we recognize 13 species in Glycyrrhiza: Glycyrrhiza acanthocarpa, Glycyrrhiza astragalina, Glycyrrhiza bucharica, Glycyrrhiza echinata, Glycyrrhiza foetida, Glycyrrhiza glabra, Glycyrrhiza gontscharovii, Glycyrrhiza lepidota, Glycyrrhiza macedonica, Glycyrrhiza pallidiflora, Glycyrrhiza squamulosa, Glycyrrhiza triphylla, and Glycyrrhiza yunnanensis. We propose a broader G. glabra that includes former Glycyrrhiza aspera, G. glabra s.s., Glycyrrhiza inflata, and Glycyrrhiza uralensis, and represents the glycyrrhizin-contained medicinal group. Our ancestral state inferences show the ancestor of Glycyrrhiza lacked glycyrrhizin, and the presence of glycyrrhizin evolved twice within Glycyrrhiza during the last one million years. Our integrative phylogenomics-MLA study not only provides new insights into long-standing taxonomic controversies of Glycyrrhizeae, but also represents a useful approach for future taxonomic studies on other plant taxa.     

Isolation and characterization of porcine parathyroid cathepsin B.

Cathepsin B was isolated from porcine parathyroid tissue and from liver by a procedure involving acetone precipitation, gel filtration, and carboxymethylcellulose chromatography. The final preparations of each migrated as single bands upon sodium dodecyl sulfate polyacrylamide gels but exhibited several minor active variants upon isoelectric focusing. The parathyroid and liver enzymes were similar to each other and also resembled cathepsin B from other sources. The molecular weights for the porcine enzymes were estimated as 25,000, and the isoelectric point was at pH 4.8. The parathyroid enzyme cleaved benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide at pH 5.8 and 37 degrees C with a Km of 0.14 mM and a kcat of 68 s-1. The pH optimum for this reaction was pH 6 to 7. The enzyme was unstable above pH 7.5 and below pH 4.5. It was strongly inhibited by HgCl2, ZnSO4, iodoacetate, iodoacetamide, and N-ethylmaleimide which indicated that it is a thiol protease, and by leupeptin, a strong inhibitor of cathepsin B from other sources. Antibodies to the parathyroid enzyme were elicited in rabbits. The antisera formed single precipitin bands upon double diffusion in agar gels against both the parathyroid and liver enzymes. Precipitin bands were formed at both pH 6 and pH 8.5 which indicated that the antisera recognized both native and denatured forms of the enzymes.     

Histone modifications in simian virus 40 and in nucleoprotein complexes containing supercoiled viral DNA.

Simian virus (SV40) nucleoprotein complexes containing circular supercoiled viral DNA were extracted from infected cells and purified by differential centrifugation. The protein content of these complexes was compared by electrophoresis on 15% acrylamide gels with the protein content of purified SV40 virions and with histones from virus-infected cells. The electrophoretic patterns of histones from each of the sources revealed several major differences. SV40 virions contained histones H3, H2B, H2A, and H4 but not H1. Nucleoprotein complexes and host cells contained all five major histone groups. Relative to cellular histones, virion and nucleoprotein complex histones were enriched 15 to 40% in histones H3 and H4. In addition to the major classes of histones, several subfractions of histones H1, H3, and H4 were observed in acrylamide gels of proteins from SV40 virions and viral nucleoprotein complexes. Acetate labeling experiments indicated that each subfraction of histones H3 and H4 had a different level of acetylation. The histones from SV40 virions and nucleoprotein complexes were acetylated to significantly higher levels than those of infected host cells. No apparent differences in phosphorylation of the major histone groups were observed.     

Computer simulation of diffusely-connected neuronal populations

This paper describes computer simulations of diffusely-connected neuronal populations. Main findings are that diffuse monosynaptic linkages between populations are selectively sensitive to synchronized clusters of action potentials in the pre-synaptic population; that diffusely-connected excitatory recurrent collaterals tend to produce rhythmic series of synchronized clusters; and that diffusely-connected inhibition (both recurrent and afferent) tend to reduce the number of cells participating in a given synchronized cluster and thereby the overall transfer rate. However, recurrent inhibition tends to increase the rate at which synchronized clusters are produced by recurrent excitation. These results suggest the speculation that diffusely connected neuronal populations are particularly prone to deal with synchronized clusters of action potentials.This work has been supported by Grant GB 33687 of the National Science Foundation, Grant 1-R01-NS-10781-01 COM of the National Institutes of Health, and by a fellowship from Zonta, International     

Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.     

Meristic variation among world hatchery stocks of rainbow trout,Salmo gairdneri Richardson

Synopsis Sixteen meristic characters were examined among 10 stocks of rainbow trout to establish their inter- and intrastock variability and relative discriminating power. The number of vertebrae, dorsal fin rays, scales above and below and lateral line, oblique scale rows and pectoral fin rays were found to be the most useful meristic characters in identifying stocks. Examination of the extent of meristic divergence among 10 hatchery or naturalized stocks from various regions of the world, using analysis of variance, the generalized distance function and per cent overlap, revealed that each stock exhibited highly significant differences in at least one meristic character, thereby demonstrating a high degree of intraspecific variability in meristic expression.In multivariate terms, stocks from Idaho and Alaska were almost totally divergent from all other stocks examined. Multivariate analysis of meristic features proves to be a useful method for identifying the extent of phenotypic variability among and within divergent stocks of rainbow trout. The observed meristic variability in test stocks of hatchery fish is seemingly genetically set within the limits previously described for native populations of rainbow trout.     

Evidence of genetic and environmental influences on meristic variation in the rainbow trout,Salmo gairdneri Richardson

Synopsis An investigation of the effect of parentage and temperature on the expression of selected meristic characters of rainbow trout, Salmo gairdneri, reveals statistical differences in the number of vertebrae, dorsal fin rays and anal fin rays among test batches of progeny of different parentage. Batches of fertilized ova of the same stock or parentage, when incubated at 9.5 °C and 14.8 °C, produced lower mean dorsal and anal fin ray counts and a higher mean pectoral fin ray count at the higher temperature. Incubation temperature had no significant effect on the number of vertebrae.Significant statistical differences occurring in various characters among groups of rainbow trout of different parental origin provide convincing evidence of genetic inputs into meristic expression. However, the existence of both environmental and genetic inputs into meristic expression in rainbow trout necessitates further investigation before the meaning of observed meristic plasticity in rainbow trout can be resolved.     

Asymmetric distribution of nitrate reductase subunits in the cytoplasmic membrane of Escherichia coli: evidence derived from surface labeling studies with transglutaminase.

The enzyme transglutaminase has been used to label surface proteins of Escherichia coli cytoplasmic membranes by covalently attaching to them a small fluorescent primary amine, dansyl cadaverine. Spheroplasts lacking outer membrane, osmotically lysed vesicles from the spheroplasts, and vesicles made by breaking cells in a French pressure cell were each labeled with transglutaminase and dansyl cadaverine. When the total cytoplasmic membrane proteins of each were examined on sodium dodecyl sulfate gels, three rather different labeling patterns were obtained. Labeling of the respiratory enzyme, nitrate reductase, in the membranes of each of these preparations was also examined. Membrane-bound nitrate reductase contains three subunits: A, B, and C. Dansyl cadaverine labeling of nitrate reductase in the presence of Triton X-100 indicated that subunits A and C could be labeled. When nitrate reductase was isolated from dansyl cadaverine-labeled spheroplasts, none of the subunits was labeled. When nitrate reductase was isolated from French press vesicles, subunit A was labeled and labeling was enhanced by the presence of nitrate during labeling. When nitrate reductase from osmotic vesicles was examined, subunit A was labeled in the presence of nitrate but no labeled subunits appeared when the vesicles were labeled in the absence of nitrate. It was concluded that (i) nitrate reductase is buried in the membrane with subunit A exposed only on the inner surface of the membrane, (ii) subunit C is sufficiently buried within the membrane so that it is inaccessible to transglutaminase, (iii) subunit B is not labeled under any condition, so its location is not known, and (iv) large osmotic vesicles are probably mosaics in which some protein components have been reoriented.     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.