Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.     

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.     

Transplacental transmission of BK virus in human.

The prevalance and distribution of BK virus antibody in women during pregnancy and the occurrence of transplacental transmission of BK virus was determined by measurement of IgM antibody in the serum. Sera were collected from 63 nonpregnant women, 71 women who had experienced spontaneous abortion, 80 in the first trimester of pregnancy and the same 80 at delivery. Umbilical cord blood was also taken at delivery. Hemagglutination inhibition (HI) tests for BK virus used the micromethod of Gardner. Results indicate that a significant level of HI antibody was present in 70% of sera from all 4 experimental groups. This showed that BK virus infection was not limited to cases of spontaneous abortion. Of the 80 pregnant mothers, 6 showed a 4-fold or greater HI antibody seroconversion to BK virus after delivery. Of these 6 seroconversion patients, sensitive antibody was detected in 3 umbilical cords. Umbilical cords of those without seroconversion had no sensitive antibody. As evidenced by 2-NE-sensitive antibody, BK virus infections were also recognized in 6 of 71 women who aborted, 4 of 80 in the first trimester of pregnancy and 2 collected after delivery. The 2-ME-sensitive antibody was not found in any of 63 samples from nonpregnant women. Data indicate that 2-ME-sensitive antibody was present only in sera of women during pregnancy and after abortion. It may be possible that BK virus persists in a latent form in many healthy women and becomes activated during pregnancy.     

Systematic notes on Anopheles Meigen (Diptera: Culicidae) species in the state of Amapá, Brazil

Identification of Anopheles nuneztovari Gabaldón and An. goeldii Rozeboom and Gabaldón based on the male genitalia traits is discussed. An. goeldii is in the synonymy of An. nuneztovari, however, characters of the aedeagus of male genitalia distinguish both species. We hypothesize that An. goeldii may be a valid species, however, further studies using molecular characters, especially ITS2 rDNA sequences will be necessary to elucidate the taxonomic status of the species. An. konderi Galv?o and Damasceno and An. forattinii Wilkerson and Sallum are registered for the first time in the state of Amapá.     

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.     

Holocentric chromosomes of Luzula elegans are characterized by a longitudinal centromere groove, chromosome bending, and a terminal nucleolus organizer region

The structure of holocentric chromosomes was analyzed in mitotic cells of Luzula elegans. Light and scanning electron microscopy observations provided evidence for the existence of a longitudinal groove along each sister chromatid. The centromere-specific histone H3 variant, CENH3, colocalized with this groove and with microtubule attachment sites. The terminal chromosomal regions were CENH3-negative. During metaphase to anaphase transition, L. elegans chromosomes typically curved to a sickle-like shape, a process that is likely to be influenced by the pulling forces of microtubules along the holocentric axis towards the corresponding microtubule organizing regions. A single pair of 45S rDNA sites, situated distal to Arabidopsis-telomere repeats, was observed at the terminal region of one chromosome pair. We suggest that the 45S rDNA position in distal centromere-free regions could be required to ensure chromosome stability.     

Simultaneous color‐coded imaging to distinguish cancer “stem‐like” and non‐stem cells in the same tumor

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133⁺ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133^? Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133⁺ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133⁺ cells were labeled with GFP and CD133^? cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010. © 2010 Wiley‐Liss, Inc.