Mapping of the rat liver endothelial membrane with lectins and glycosylated ferritins

We explored the luminal surface of liver sinus endothelium for the presence of lectin receptors and lectinlike substances capable of interacting with specific sugars. We used ferritin-conjugated lectins and glycosylated ferritins as probes. Incubation of small blocks of rat liver with these probes led to the binding of concanavalin A (on A), Ricinus communis (RCA), wheat germ agglutinin (WGA), phytohemagglutinin (PHA) and mannosyl ferritins to the luminal surface of endothelium. Ulex europaeus agglutinin I (UEA), fucosyl, galactosyl, and chitobiosyl-ferritins did not bind. The binding was patchy and sparse in the case of Con A and mannosyl-ferritins but uniform for others. Binding density did not correlate with hemagglutinability of lectins, suggesting that the difference in the hemagglutinability of these lectins did not account for the difference in their binding densities. Bindings were all completely inhibited in the presence of excess specific sugar inhibitors, indicating the specificity of binding. The distribution of binding was segregated on the endothelial membrane, being heaviest on luminal pits. To define the functional significance of this segregated distribution, sinus endothelium was compared to portal-vein endothelium in which endothelial fenestrations are also seen; and these fenestrations as well as pits may be covered by a thin diaphragm. Of interest was the total absence of binding to the diaphragm. The significance of these findings is discussed.     

Transstadial Transmission of Borrelia turcica in Hyalomma aegyptium Ticks

Borrelia turcica comprises the third major group of arthropod-transmitted borreliae and is phylogenetically divergent from other Borrelia groups. The novel group of Borrelia was initially isolated from Hyalomma aegyptium ticks in Turkey and it was recently found in blood and multiple organs of tortoises exported from Jordan to Japan. However, the ecology of these spirochetes and their development in ticks or the vertebrate hosts were not investigated in detail; our aims were to isolate the pathogen and to evaluate the possibility of transstadial transmission of Borrelia turcica by H. aegyptium ticks. Ticks were collected from Testudo graeca tortoises during the summer of 2013 from southeastern Romania. Engorged nymphs were successfully molted to the adult stage. Alive B. turcica was isolated from molted ticks by using Barbour-Stoenner-Kelly (BSK) II medium. Four pure cultures of spirochetes were obtained and analyzed by PCR and sequencing. Sequence analysis of glpQ, gyrB and flaB revealed 98%–100% similarities with B. turcica. H. aegyptium ticks collected from T. graeca tortoises were able to pass the infection with B. turcica via transstadial route, suggesting its vectorial capacity.     

A functional assay for microRNA target identification and validation

MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.     

Design, synthesis, and biological evaluation of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase

A series of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues lacking the benzoylglutamate subunit were prepared and examined as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase).     

Liver endothelium mediates the uptake of iron-transferrin complex by hepatocytes

We have previously shown that in the liver, transferrin (TF) receptors are limited to endothelial cells, and hepatocytes and Kupffer cells do not have TF receptors. To study the transport of iron into hepatocytes, we fractionated liver cell suspensions into endothelium and hepatocyte fractions. At 4 degrees C liver (but not umbilical cord) endothelium bound Fe-TF with a saturable kinetics. At 37 degrees C, the endothelial uptake was followed by its gradual release. Transendothelial transport of TF was visually demonstrated by perfusion of liver using colloidal gold-labeled TF. The released Fe-TF acquired the potential for binding to fresh target hepatocytes and binding was not inhibited by excess cold TF but was inhibitable by asialofetuin, suggesting galactosyl receptors and not TF receptors as a recognition mechanism. Isoelectrofocusing of the supernate after preincubation for 90 min at 37 degrees C with endothelial cells, demonstrated the presence of a newly generated band which co-migrated with asialotransferrin. We conclude that Fe-TF is initially removed by liver endothelium where it is modified probably by desialation to expose the galactosyl residues of the glycoproteins. The modified molecule is subsequently released and recognized by hepatocytes through a TF receptor-independent mechanism which may involve galactosyl receptors of hepatocytes. The findings indicate a key role for endothelium in the transport of Fe-TF into the liver and may suggest a physiological function for galactosyl receptors on hepatocyte surface.     

Ceruloplasmin receptors in liver cell suspensions are limited to the endothelium

The interaction of ceruloplasmin (CP) with isolated liver cell suspensions was studied using 125I-labeled and latex minibead-derivatized CP. Fractionation of liver cell suspensions was done using metrizamide gradient centrifugation. In crude liver cell suspensions only endothelial cells, but not hepatocytes and Kupffer cells bound the minibead probe. The binding was specific and inhibited by excess native CP. These results were confirmed using 125I-CP combined with cell fractionation technique. Kinetic data, obtained from the latter system, indicated a dissociation constant (Kd) of 1 X 10(-7) M and the number of receptors to be 5.7 X 10(5) per endothelial cell. The exclusive binding of CP to liver endothelium suggests that this cell may mediate the hepatocytes uptake of CP and is, therefore, a crucial element of the tissue-blood barrier.     

Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage.

The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage. In addition, it has a very high degree of chromosome loss and/or nondisjunction. We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S. pombe rad13/human XPG family. Using degenerate PCR, we have cloned the human homolog of the rad2 gene. Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant. We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage. Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms.