Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.     

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.     

Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life

MDC9 (ADAM9/meltrin gamma) is a widely expressed and catalytically active metalloprotease-disintegrin protein that has been implicated in the ectodomain cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and as an alpha secretase for the amyloid precursor protein. In this study, we evaluated the expression of MDC9 during development and generated mice lacking MDC9 (mdc9(-/-) mice) to learn more about the function of this protein during development and in adults. During mouse development, MDC9 mRNA is ubiquitously expressed, with particularly high expression levels in the developing mesenchyme, heart and brain. Despite the ubiquitous expression of MDC9, mdc9(-/-) mice appear to develop normally, are viable and fertile, and do not have any major pathological phenotypes compared to wild-type mice. Constitutive and stimulated ectodomain shedding of HB-EGF is comparable in embryonic fibroblasts isolated from mdc9(-/-) and wild-type mice, arguing against an essential role of MDC9 in HB-EGF shedding in these cells. Furthermore, there were no differences in the production of the APP alpha and gamma secretase cleavage product (p3) and of beta- and gamma-secretase cleavage product (A beta) in cultured hippocampal neurons from mdc9(-/-) or wild-type mice, arguing against an essential major role of MDC9 as an alpha-secretase in mice. Further studies, including functional challenges and an evaluation of potential compensation by, or redundancy with, other members of the ADAM family or perhaps even with other molecules will be necessary to uncover physiologically relevant functions for MDC9 in mice.     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy–immunogold labeling

The lignification process in mature Norway spruce [Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)–immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.     

The Arabidopsis SKP1-like genes present a spectrum of expression profiles

The yeast Skp1 protein is a component of the SCF complex, an E3 enzyme involved in the specific protein degradation pathway via ubiquitination. Skp1 binds to F-box proteins to trigger specific recognition of proteins targeted for degradation. SKP1-like genes have been found in a variety of eukaryotes including yeast, man, Caenorhabditis elegans and Arabidopsis thaliana. The Arabidopsis genome contains 20 SKP1-like genes called ASK (for Arabidopsis SKP1-like), among which only ASK1 has been characterized in detail. The analysis of the expression pattern of the ASK genes in Arabidopsis should provide key information for the understanding of the biological role of this family in protein degradation and in different cellular mechanisms. In this paper, we describe the expression profiles of 19 ASK promoter-GUS fusions in stable transformants of Arabidopsis, with a special emphasis on floral organ development. Four ASK promoters did not show any detectable expression in either inflorescences or seedlings. Our results on the ASK1 expression profile are consistent with previous reports. Several ASK promoters show clear tissue-specific expression (for instance in the connective of anthers or in the embryo). We also found that almost half (9/19) of ASK promoters direct a post-meiotic expression in the male gametophyte. Tight regulation of the expression of this gene family indicates a crucial role of the ubiquitin degradation pathway during development, particularly during male gametophyte development.     

Genetic interactions between FLM and other flowering-time genes in Arabidopsis thaliana

FLOWERING LOCUS M (FLM) is a MADS-domain gene that acts as an inhibitor of flowering in Arabidopsis. Here we describe the genetic interaction of FLM with genes in the photoperiod and autonomous flowering pathways. Although the sequence of FLM is most similar to that of FLC, FLM and FLC interact with different flowering pathways. It has been previously shown that flc lesions suppress the late-flowering phenotype of FRI-containing lines and autonomous-pathway mutants. However, flm lesions suppress the late-flowering phenotype of photoperiod-pathway mutants but not that of FRI-containing lines or autonomous-pathway mutants. Another MADS-domain flowering repressor with a mutant phenotype similar to FLM is SVP. The late-flowering phenotype of FLM over-expression is suppressed by the svp mutation, and an svp flm double mutant behaves like the single mutants. Thus FLM and SVP are in the same flowering pathway which interacts with the photoperiod pathway. Abbreviations: CO, CONSTANS; FLC, FLOWERING LOCUS C; FLM, FLOWERING LOCUS M; FRI, FRIGIDA; GI, GIGANTEA; LD, LUMINIDEPENDENS; SVP, SHORT VEGETATIVE PHASE; FCA is not an abbreviation     

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.     

Potential role for ADAM15 in pathological neovascularization in mice

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.     

Seed lectin from pisum arvense: isolation, biochemical characterization and amino acid sequence

A glucose/mannose lectin was purified by affinity chromatography from Pisum arvense seeds (PAL) and the 50 kDa molecular mass in solution determined by size exclusion chromatography. SDS-PAGE and electrospray ionization mass spectrometry showed two distinct polypeptide chains: alpha (Mr. 5591 Da) and beta (19986 Da). The lectin was extensively characterized in terms of its biochemical and biological aspects. The amino acid sequence was established by Edman degradation of overlapping peptides. PAL in solution behaves as a dimer and has its monomeric structure formed by two distinct polypeptide chains named alpha (Mr. 5591 Da) and beta (19986 Da) by Electrospray ionization (ESI) mass spectrometry. PAL possesses identical amino acid sequences to that of pea seed lectin but undoubtedly does not exhibit sequence heterogeneity. It is discussed that P. arvense should be considered as a synonym of P. sativum. Furthermore, like pea lectin, PAL discriminates biantennary fucosylated glycan, determined by surface plasmon resonance.