Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

The effect of salts on the kinetics of iron release from N-terminal and C-terminal monoferrictransferrins

The kinetics of iron release from N-terminal and C-terminal monoferric human transferrins has been studied using EDTA as the accepting chelate. In the absence of added salts iron release from the N-terminal site is more facile but the relative lability can be reversed by the addition of NaClO₄, NaCl and LiCl. The results indicate that both anions and cations can affect the lability of the two sites. Since the relative lability of the two monoferrictransferrins is affected by fairly moderate concentrations of NaCl and NaClO₄ we suggest that the ionic composition serum may play an important role in determining the observed distribution of iron among the sites. A new method for the preparation of N-terminal monoferrictransferrin is described.     

Identification and cloning of two histone fold motif-containing subunits of HeLa DNA polymerase epsilon

HeLa DNA polymerase epsilon (pol epsilon), possibly involved in both DNA replication and DNA repair, was previously isolated as a complex of a 261-kDa catalytic subunit and a tightly bound 59-kDa accessory protein. Saccharomyces cerevisiae pol epsilon, however, consists of four subunits: a 256-kDa catalytic subunit with 39% identity to HeLa pol epsilon p261, a 80-kDa subunit (DPB2) with 26% identity to HeLa pol epsilon p59, a 23-kDa subunit (DPB3), and a 22-kDa subunit (DPB4). We report here the identification and the cloning of two additional subunits of HeLa pol epsilon, p17, and p12. Both proteins contain histone fold motifs which are present also in S. cerevisiae DPB4 and DPB3. The histone fold motifs of p17 and DPB4 are related to that of subunit A of the CCAAT binding factor, whereas the histone fold motifs found in p12 and DPB3 are homologous to that in subunit C of CCAAT binding factor. p17 together with p12, but not p17 or p12 alone, interact with both p261 and p59 subunits of HeLa pol epsilon. The genes for p17 and p12 can be assigned to chromosome locations 9q33 and 2p12, respectively.     

Application of beta-irradiation through the struts of a previously deployed stent

The application of beta-radiation in coronary arteries is a promising new technique for the treatment of in-stent restenosis. This is the first case in which the 5 F. delivery catheter of the Beta-Cath trade mark system was advanced through the struts of a stent, previously deployed in an adjacent branch, so as to deliver radiation to the target vessel.     

Identification to the species level of <Emphasis Type="Italic">Lactobacillus</Emphasis> isolated in probiotic prospecting studies of human,animal or food origin by 16S-23S rRNA restriction profiling

Background  

The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling.     

Morphological differences among egg nests and adult individuals of Cicadatra persica (Hemiptera,Cicadidae), distributed in Erneh,Syria

The aim of this study is determining the different patterns of egg nests and the morphological differences between the specimens of Cicadatra persica Kirkalidy, 1909 (Hemiptera: Cicadidae) distributed in fruit orchards in Erneh located on AL-Sheikh mountain south west of Syria. The appearance of 80 egg nests was studied, and the results showed that there were two basic patterns of egg nests laid by Cicadatra persica, 90% of the egg nests were of the first pattern (consists of several adjacent slits), while 10% of them were of the second pattern (consists of several divergent slits). A random sample consisting of 300 specimens (150 males and 150 females) were also studied concentrating on the differences in the color of the supra-antennal plate and in the number of spurs on the tibia of the hind legs. The results showed that there were two basic patterns of individuals based on the differences in the color of supra-antennal plate. The first pattern (individuals with yellow supra-antennal plates), constituted more than 90%, and the second one (individuals with black supra-antennal plates) constituted less than 10%. The results also showed that there were 27 different patterns based on the number of spurs on the tibia of the hind legs. One of them was a common pattern (2, 3) whose individuals have 2 spurs on the upper side of the tibia of the hind legs and 3 spurs on the lateral side of the tibia of the hind legs. The total percent of this common pattern was 76%. The other 26 patterns were different from each other, and the total percent of all these different patterns was 24%.     

Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.     

Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase epsilon

To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo.