Isolation and Characterization of a Cytosolic Phospholipase A2 from Bovine Adrenal Medulla

Abstract: We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A₂ (PLA₂), which is localized in the cytosol. In the present study, this PLA₂ was purified 1,097-fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20–1,000 µ M Ca²⁺ and has a pH optimum near 8.0. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA₂ is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X-100 (0.01%) and deoxycholate (1 m M ) but inhibited at higher concentrations (0.1% and 3 m M , respectively) of these detergents. Furthermore, heat treatment (57°C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity. p -Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA₂, has little effect until 100 µ M , and 2–10 m M dithiothreitol totally inactivated the enzyme. The purified PLA₂ displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn -2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA₂, which was isolated and characterized in this study, represents a type of PLA₂ that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA₂ will help to reveal whether the enzyme is important for exocytosis.     

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo

Background

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.

Methods and results

We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino‐sugar‐based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin‐cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.

Conclusions

These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Zinc tolerance and hyperaccumulation are genetically independent characters

The hyperaccumulation of metals by a rare class of plants is a fascinating and little understood phenomenon. No genetic analysis has been possible since no intraspecific variation is known for this character. Here, we report on crosses between the zinc-hyperaccumulating and -tolerant species Arabidopsis halleri and the non-hyperaccumulating, non-tolerant species Arabidopsis petraea. The F2 segregates for both characters and it appears that the two characters are genetically independent. The data for tolerance are consistent with a single major gene for this character (although the number of genes for hyperaccumulation cannot be determined), and is probably not very large.     

Multilevel Control of Organelle DNA Sequence Length in Plants

We have compared the length of noncoding organelle DNA spacers in a broad sample of plant species characterized by different life history traits to test hypotheses regarding the nature of the mechanisms driving changes in their size. We first demonstrate that the spacers do not evolve at random in size but have experienced directional evolutionary trends during plant diversification. We then study the relationships between spacer lengths and other molecular features and various species attributes by taking into account population genetic processes acting within cell lineages. Comparative techniques are used to test these relationships while controlling for species phylogenetic relatedness. The results indicate that spacer length depends on mode of organelle transmission, on population genetic structure, on nucleotide content, on rates of molecular evolution, and on life history traits, in conformity with predictions based on a model of intracellular competition among replicating organelle genomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distinct male reproductive strategies in two closely related oak species

Reproductive strategies of closely related species distributed along successional gradients should differ as a consequence of the trade‐off between competition and colonization abilities. We compared male reproductive strategies of Quercus robur and Q. petraea, two partly interfertile European oak species with different successional status. In the studied even‐aged stand, trees of the late‐successional species (Q. petraea) grew faster and suffered less from intertree competition than trees of the early‐successional species (Q. robur). A large‐scale paternity study and a spatially explicit individual‐based mating model were used to estimate parameters of pollen production and dispersal as well as sexual barriers between species. Male fecundity was found to be dependent both on a tree's circumference and on its environment, particularly so for Q. petraea. Pollen dispersal was greater and more isotropic in Q. robur than in Q. petraea. Premating barriers to hybridization were strong in both species, but more so in Q. petraea than in Q. robur. Hence, predictions based on the competition–colonization trade‐off are well supported, whereas the sexual barriers themselves seem to be shaped by colonization dynamics.     

A novel locus for autosomal dominant nonsyndromic hearing loss (DFNA44) maps to chromosome 3q28–29

Hereditary non-syndromic sensorineural hearing loss (NSSHL) is a genetically highly heterogeneous group of disorders. Autosomal dominant forms account for up to 20% of cases. To date, 39 loci have been identified by linkage analysis of affected families that segregate NSSHL forms in the autosomal dominant mode (DFNA). Investigation of a large Spanish pedigree with autosomal dominant inheritance of bilateral and progressive NSSHL of postlingual onset excluded linkage to known DFNA loci and, in a subsequent genome-wide scan, the disorder locus was mapped to 3q28-29. A maximum two-point LOD score of 4.36 at theta=0 was obtained for marker D3S1601. Haplotype analysis placed the novel locus, DFNA44, within a 3-cM genetic interval defined by markers D3S1314 and D3S2418. Heteroduplex analysis and DNA sequencing of coding regions and exon/intron boundaries of two genes (CLDN16 and FGF12) in this interval did not reveal disease-causing mutations.     

Mitochondria-derived glutamate at the interplay between branched-chain amino acid and glucose-induced insulin secretion

In pancreatic beta-cells, glutamate has been proposed to mediate insulin secretion as a glucose-derived factor, although it is also considered for its sole catabolic function. Hence, changes in cellular glutamate levels are a matter of debate. Here, we investigated the effects of glucose and the glutamate precursor glutamine on kinetics of glutamate levels together with insulin secretion in INS-1E beta-cells. Preincubation at low (1 mM) glucose resulted in reduced cellular glutamate levels, which were doubled by exposure to glutamine. In glutamine-deprived cells, 5 mM glucose restored glutamate concentrations. Incubation at 15 mM glucose increased cellular glutamate, along with stimulation of insulin secretion, following both glutamine-free and glutamine-rich preincubations. Nuclear magnetic resonance (NMR) spectroscopy of INS-1E cells exposed to 15 mM D-[1-(13)C]glucose revealed glutamate as the major glucose metabolic product. Branched-chain amino acids, such as leucine, reduced cellular glutamate levels at low and intermediate glucose. This study demonstrates that glucose stimulates glutamate generation, whereas branched-chain amino acids promote competitive glutamate expenditure.     

Seasonal fluctuations and long-term persistence of pathogenic populations of Agrobacterium spp. in soils

Short- and long-term persistence of pathogenic (i.e., tumor forming) agrobacteria in soil was investigated in six nursery plots with a history of high crown gall incidence. No pathogenic Agrobacterium strains were isolated in soil samples taken in fall and winter in any plots, but such strains were isolated from both bulk soils and weed rhizospheres (over 0.5 x 10(5) pathogenic CFU/g of bulk soil or rhizosphere) in three out of six plots in spring and summer. PCR amplifications of a vir sequence from DNA extracted from soil confirmed the presence of Ti plasmids in summer and their absence in fall and winter. The results indicate that strains that harbor a Ti plasmid had an unforeseen positive fitness versus Ti plasmid-free strains in soil and rhizosphere in spring and summer in spite of the apparent absence of tumor, and hence of opines. The gain of fitness occurred during a bloom of all cultivable agrobacteria observed only in conducive soils. An evolution of the pathogenic population was recorded during a 4-year period in one particularly conducive soil. In 1990, the pathogenic population in this soil consisted of only biovar 1 strains harboring both octopine- and nopaline-type Ti plasmids. In 1994, it consisted of only nopaline-type Ti plasmids equally distributed among biovar 1 and 2 strains. These results suggest that nopaline-type Ti plasmids conferred a better survival ability than octopine-type Ti plasmids to biovar 2 agrobacteria under the present field conditions.     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.