Influence of sialic acid on cell surface properties in I-cell disease fibroblasts

Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing. This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1, National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health.     

Hairy root induction of<Emphasis Type="Italic"> Papaver somniferum</Emphasis> var.<Emphasis Type="Italic"> album</Emphasis>, a difficult-to-transform plant,by<Emphasis Type="Italic"> A. rhizogenes</Emphasis> LBA 9402

Two strains of Agrobacterium rhizogenes (15834, LBA 9402) and one Agrobacterium tumefaciens strain [GV 3101 (PMP90RK, p35SGUS-2)] and four culture media were tested and compared for their ability to induce hairy root formation on wounded Papaver somniferum L. hypocotyls. Five weeks after the infection with A. rhizogenes LBA 9402, hairy roots appeared on 80% of the hypocotyls maintained in the hormone-free liquid medium. Six hairy-root cultures were established. Transformation was confirmed by polymerase chain reaction analysis. One clone was analysed for its alkaloid production. The total alkaloid content was higher in the transformed roots (0.46±0.06% DW) than in the untransformed roots (0.32±0.05% DW). The transformed roots accumulated three times more codeine (0.18±0.02% DW) than intact roots (0.05±0% DW). Moreover, morphine (0.255±0.03% DW) and sanguinarine (0.014±0% DW) were found in the liquid culture medium.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - LS Linsmaier and Skoog     

High- and low-uptake forms of β-hexosaminidase in human platelets Selective retention of the high-uptake form during stimulation with thrombin

Human platelets are rich in β-hexosaminidase and other acid hydrolases contained in organelles (lysosomes) distinct from α-granules and dense granules. Incubation of platelets with bovine or human thrombin (100 U/ml for 5 min at 37°C) induces the secretion of 100% of the contents of α- and dense granules, but only 40–60% of total β-hexosaminidase from lysosomes. Both isozymes Hex A and Hex B are secreted in the same proportion as found intracellulary. There is no selective recapture or plasma membrane binding by platelets of secreted β-hexosaminidase. The secreted enzyme is of the low-uptake type, i.e., it is poorly recognized by the phosphomannosyl receptor-mediated uptake mechanism of fibroblasts, while the retained enzyme is a 3-fold higher uptake form. Preincubation of platelets with NH₄Cl (10 mM, 2 h), followed by thrombin stimulation, results in secretion of all β-hexosaminidase as a low-uptake form. The data support the hypothesis that there are secretory and nonsecretory forms of lysosomes. The secretory lysosomes would contain low-uptake forms of hydrolases in addition to acid phosphatase, while the nonsecretory lysosomes would contain high-uptake hydrolases and be acid phosphatase-deficient. Conditions where the contents of both lysosomal populations were released together, i.e., amine treatment followed by thrombin induction, or extraction of unstimulated cells, would result in the exposure of high-uptake phosphomannosylated hydrolases released from one population of lysosomes to acid phosphatase released from the second population of lysosomes with their subsequent conversion to low-uptake forms.     

Validation of multi-stage telephone-based identification of cognitive impairment and dementia

Background  

Many types of research on dementia and cognitive impairment require large sample sizes. Detailed in-person assessment using batteries of neuropyschologic testing is expensive. This study evaluates whether a brief telephone cognitive assessment strategy can reliably classify cognitive status when compared to an in-person "gold-standard" clinical assessment.     

The <Emphasis Type="Italic">Arabidopsis</Emphasis> LHP1 protein is a component of euchromatin

The HP1 family proteins are involved in several aspects of chromatin function and regulation in Drosophila, mammals and the fission yeast. Here we investigate the localization of LHP1, the unique Arabidopsis thaliana HP1 homolog known at present time, to approach its function. A functional LHP1–GFP fusion protein, able to restore the wild-type phenotype in the lhp1 mutant, was used to analyze the subnuclear distribution of LHP1 in both A. thaliana and Nicotiana tabacum. In A. thaliana interphase nuclei, LHP1 was predominantly located outside the heterochromatic chromocenters. No major aberrations were observed in heterochromatin content or chromocenter organization in lhp1 plants. These data indicate that LHP1 is mainly involved in euchromatin organization in A. thaliana. In tobacco BY-2 cells, the LHP1 distribution, although in foci, slightly differed suggesting that LHP1 localization is determined by the underlying genome organization of plant species. Truncated LHP1 proteins expressed in vivo allowed us to determine the function of the different segments in the localization. The in foci distribution is dependent on the presence of the two chromo domains, whereas the hinge region has some nucleolus-targeting properties. Furthermore, like the animal HP1β and HP1γ subtypes, LHP1 dissociates from chromosomes during mitosis. In transgenic plants expressing the LHP1–GFP fusion protein, two major localization patterns were observed according to cell types suggesting that localization evolves with age or differentiation states. Our results show conversed characteristics of the A. thaliana HP1 homolog with the mammal HP1γ isoform, besides specific plant properties.     

<Emphasis Type="Italic">In vitro</Emphasis> collecting (IVC). I. The effect of collecting method and antimicrobial agents on contamination in temperate and tropical collections

Summary In vitro collecting is the process of initiating tissue cultures in the field. In order for in vitro collecting to be broadly available as a technique for collecting plant germplasm, the levels of contamination in such cultures must be controlled. Two techniques for in vitro collecting were compared: leaf punch and needle collecting. The effectiveness of these methods for collecting leaf and stem tissues from plants at tropical and temperature sites was compared. Stem tissue collected by the needle collecting method gave cultures with an average contamination percentage of 31% and 16%, from the tropical and temperate sites, respectively, while with the leaf punch method, average contamination percentages were 90% and 69%. The effectiveness of antimicrobial agents in reducing contamination in leaf punch cultures was evaluated. Addition of the fungicide benlate and the antibiotics cefotaxime and vancomycin, to the leaf punch collections reduced contamination to an average of 30% in the tropical collections and 35% in the temperate collections. Over 90% of both tropical and temperature species collected in multiple samples of 10 or more had at least one clean sample using this medium. The use of either the leaf punch method in combination with a fungicide and antibiotics or the needle collecting technique yielded a high percentage of clean tissues for study and growth.