Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12.

The normal human granulocyte vitamin B12-binding protein, transcobalamin I, and transcobalamin III, have been labeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate and utilized for plasma clearance studies performed with rabbits. Both moieties of 125I-labeled granulocyte vitamin B12-binding protein-[57Co]vitamin B12 were cleared rapidly from the plasma (is less than 90% by 5 min) by the liver. After 30 min, the bulk of the 125I reappeared in the plasma in small molecular weight (less than 1000) form and was rapidly excreted in the urine. After 60 min the bulk of the [57Co]vitamin B12 reappeared in the plasma bound to rabbit transcobalamin II and was subsequently taken up by a variety of tissues. Approximately 15% of the 125I-labeled granulocyte vitamin B12-binding protein-[57Co-a1vitamin B12 was excreted intact into the bile during the period from 10 to 80 min after injection. The hepatic uptake of the protein-vitamin B12 complex was blocked by the prior injection of desialyzed fetuin but not by native fetuin. Similar results were obtained with 125I-labeled transcobalamin III-[57Co]vitamin B12. Approximately 90% of both moieties of 125I-labeled transcobalamin I-[57Co]vitamin B12 had prolonged plasma survivals similar to that of 125I-labeled bovine serum albumin. After treatment with neuraminadase, both moieties of the 125I-labeled transcobalamin I-[57Co]vitamin B12 complex were cleared rapidly from the plasma by the liver in a manner that was indistinguishable from that observed in the case of untreated granulocyte vitamin B12-binding protein and transcobalamin III. These observations indicate that desialyzed transcobalamin I and the native forms of the granulocyte vitamin B12-binding protein and transcobalamin III are cleared from plasma by the mechanism elucidated by Ashwell and Morell (Ashwell, G., and Morell A. G. (1974) Adv. Enzymol. 41, 99-128) that is capable of clearing a wide variety of asialoglycoproteins. These observations have implications concerning the function of the human R-type vitamin B12-binding proteins, the nature of the enterohepatic circulation of vitamin B12, the biological significance of the mechanism described by Ashwell and Morell, and the etiology of the increased plasma concentration of human R-type protein that occurs frequently in chronic myelogenous leukemia and occasionally in hepatocellular carcinoma and other solid tumors.     

Characterization of the binding of human growth hormone to microsomal membranes from rat liver.

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 X 10(10) +/- 0.2 X 10(10)M-1 and 0.03 X 10(10) +/- 0.007 X 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.     

The role of adhesion molecules and chemokine receptor CXCR4 (CD184) in small cell lung cancer

Small-cell lung cancer (SCLC) is a particularly aggressive form of lung cancer. Responsible for this highly malignant phenotype is an early and widespread metastasis with a high propensity of SCLC cells for bone marrow involvement and the ability to develop resistance against chemotherapeutic agents. Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and adhesion molecules. There is growing evidence that the chemokine stromal derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 (CD184) regulate migration and metastasis of a variety of cancers including SCLC. SCLC cells express high levels of functional CXCR4 receptors. Engagement of CXCR4 by CXCL12 leads to an upregulation of integrin-mediated adhesion in SCLC and other tumor cells. Activation of CXCR4 chemokine receptors and integrins on SCLC cells promotes adhesion to accessory cells (such as stromal cells) and extracellular matrix molecules within the tumor microenvironment. These adhesive interactions result in an increased resistance of SCLC cells to chemotherapy. As such, inhibitors of the CXCR4/CXCL12 axis and/or integrin activation may increase the chemosensitivity of SCLC cells and lead to new therapeutic avenues for patients with SCLC.     

Peptide modification by introduction ofα-trifluoromethyl substituted amino acids

Summary Methodology for the synthesis and incorporation of-trifluoromethyl substituted amino acids into N- and C-terminal position of peptides is described. The incorporation of-trifluoromethyl substituted amino acids into strategical positions of peptides enhances proteolytic stability and lipophilicity. Furthermore, it improves transport rates in vivo and permeability through certain body barriers.     

Nhs: network-based hierarchical segmentation for cryo-electron microscopy density maps

Cryo-electron microscopy (cryo-EM) experiments yield low-resolution (3-30 ?) 3D-density maps of macromolecules. These density maps are segmented to identify structurally distinct proteins, protein domains, and subunits. Such partitioning aids the inference of protein motions and guides fitting of high-resolution atomistic structures. Cryo-EM density map segmentation has traditionally required tedious and subjective manual partitioning or semisupervised computational methods, whereas validation of resulting segmentations has remained an open problem in this field. We introduce a network-based hierarchical segmentation (Nhs) method, that provides a multi-scale partitioning, reflecting local and global clustering, while requiring no user input. This approach models each map as a graph, where map voxels constitute nodes and weighted edges connect neighboring voxels. Nhs initiates Markov diffusion (or random walk) on the weighted graph. As Markov probabilities homogenize through diffusion, an intrinsic segmentation emerges. We validate the segmentations with ground-truth maps based on atomistic models. When implemented on density maps in the 2010 Cryo-EM Modeling Challenge, Nhs efficiently and objectively partitions macromolecules into structurally and functionally relevant subregions at multiple scales.     

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra-Golgi pH

Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.     

Phylogenetic systematics of Colotis and associated genera (Lepidoptera: Pieridae): evolutionary and taxonomic implications

We investigated the genetic diversity and phylogenetic placement of the butterflies in the genus Colotis and eight related pierid genera using sequence information from two mitochondrial and two nuclear genes. To establish the status of species, we initially barcoded 632 specimens representative of all genera and most species and subspecies in those genera. A subset was then selected for phylogenetic analysis where additional gene regions were sequenced: 16S rRNA (523 bp), EF‐1α (1126 bp) and wg (404 bp). DNA barcode results were largely congruent with the traditional classification of species in the Colotis group, but deep splits or lack of genetic divergence in some cases supported either species‐level differentiation or synonymy. Despite using information from four genes, the deeper nodes in our phylogeny were not strongly supported, and monophyly of the ‘Colotis group’ and the genera Colotis and Eronia could not be established. To preserve the monophyly of Colotis, we revive the genus Teracolus for three outlying species previously in Colotis (i.e. Colotis eris, Colotis subfasciatus and Colotis agoye), as well as the genus Afrodryas for Eronia leda. The position of Calopieris is unresolved although it appears to be well outside the molecular variation in Colotis (s.l.). A dispersal/vicariance analysis suggested that major diversification in Colotis (s.str.) occurred in Africa with subsequent dispersal to India and Madagascar.