Modifying dipalmitoylphosphatidylcholine monolayers by n-hexadecanol and dipalmitoylglycerol

The monolayer structure of pure dipalmitoylphosphatidylcholine (DPPC) and equimolar mixtures of DPPC/n-hexadecanol (C(16)OH) and DPPC/dipalmitoylglycerol (DPG) are studied by the film balance technique and grazing incidence X-ray diffraction measurements. At 20 degrees C, the binary systems exhibit complete miscibility. In contrast to pure DPPC monolayers, a condensing effect is observed in the presence of both non-phospholipid additives; but the phase transition behavior differs. The tilt angle of the hydrocarbon chains in the DPPC/C(16)OH mixture is significantly smaller than in pure DPPC monolayers. The tilt of the chains is even further reduced in the mixed monolayer of DPPC/DPG. A comparison of the three systems reveals distinct structural features such as phase state, chain tilt, and molecular area over a wide range of surface pressures. Therefore, these monolayers provide a highly suitable model to investigate the influence of structural parameters on biological processes occurring at the membrane surface, e.g. enzymatic reactions and adsorption events.     

Melanosome diversity and convergence in the evolution of iridescent avian feathers—Implications for paleocolor reconstruction

Some of the most varied colors in the natural world are created by iridescent nanostructures in bird feathers, formed by layers of melanin‐containing melanosomes. The morphology of melanosomes in iridescent feathers is known to vary, but the extent of this diversity, and when it evolved, is unknown. We use scanning electron microscopy to quantify the diversity of melanosome morphology in iridescent feathers from 97 extant bird species, covering 11 orders. In addition, we assess melanosome morphology in two Eocene birds, which are the stem lineages of groups that respectively exhibit hollow and flat melanosomes today. We find that iridescent feathers contain the most varied melanosome morphologies of all types of bird coloration sampled to date. Using our extended dataset, we predict iridescence in an early Eocene trogon (cf. Primotrogon) but not in the early Eocene swift Scaniacypselus, and neither exhibit the derived melanosome morphologies seen in their modern relatives. Our findings confirm that iridescence is a labile trait that has evolved convergently in several lineages extending down to paravian theropods. The dataset provides a framework to detect iridescence with more confidence in fossil taxa based on melanosome morphology.     

Mutagenesis of CXCR4 Identifies Important Domains for Human Immunodeficiency Virus Type 1 X4 Isolate Envelope-Mediated Membrane Fusion and Virus Entry and Reveals Cryptic Coreceptor Activity for R5 Isolates

CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.     

Secretion and folding of human growth hormone in Escherichia coli

Abstract

Phytoremediation is the use of plants for the treatment of environmental pollution, including chlorinated organics. although conceptually very attractive, removal and biodegradation of chlorinated pollutants by plants is a rather slow and inefficient process resulting in incomplete treatment and potential release of toxic metabolites into the environment. In order to overcome inherent limitations of plant metabolic capabilities, plants have been genetically modified, following a strategy similar to the development of transgenic crops: genes from bacteria, fungi, and mammals involved in the metabolism of organic contaminants, such as cytochrome p-450 and glutathione substrate catabolic genes, natural or engineered, for the simultaneous remediation of a range of pollutants, such as usually found in contaminated sites, e.g., chlorinated solvent, metals, and nitroaromatics. In addition, biodegradation of many xenobiotics are catalyzed by similar, broad-substrate enzymes, such as cytochrome P-450 monoxygenases, glutathione S-transferases, and fungal peroxidases, that can potentially be used for the treatment of multiple pollutants. Moreover, the introduction of multiple transgenes involved in different phases of the metabolism of xenobiotics in plants, i.e., uptake by roots and the different phases of the green liver model, would allow enhancing both the removal and metabolism of several toxic compounds and could therefore help overcome a major limitation inherent to phytoremediation, i.e., the threat that accumulated toxic compounds would volatilize or otherwise contaminate the food chain. An important barrier to the application of transgenic plants for bioremediation in the field is associated with the true or perceived risk of horizontal gene transfer to related wild or cultivated plants. Therefore, it is likely that the next generation of transgenic plants will involve systems preventing such a transfer, for instance by the introduction of transgenes into chloroplastic DNA or the use of conditional lethality genes (Davison, 2005). Since bacteria naturally exchange plasmids via conjugation, endophytes that gain genes involved in pollutant degradation might not be considered ‘genetically modified’ and may be subject to fewer restrictions in usage.     

Development of biochemical specialization and organelle partitioning in the single-cell C4 system in leaves of Borszczowia aralocaspica (Chenopodiaceae)

The terrestrial plant Borszczowia aralocaspica (Chenopodiaceae) has recently been shown to contain the entire C(4) photosynthesis mechanism within individual, structurally and biochemically polarized chlorenchyma cells rather than in a dual cell system, as has been the paradigm for this type of carbon fixation (Nature 414: 543-546, 2001). Analysis of carbon isotope composition and (14)CO(2) fixation shows that photosynthesis and growth of B. aralocaspica occurs through carbon acquired by C(4) photosynthesis. The development of this unique single-cell C(4) system in chlorenchyma cells was studied by analysis of young (0.2-0.3 cm length), intermediate (ca. 0.5-0.6 cm length), and mature leaves (ca. 3 cm length). The length of chlorenchyma cells approximately doubles from young to intermediate and again from intermediate to the mature leaf stage. In young chlorenchyma cells, there is a single type of chloroplast; the chloroplasts are evenly distributed throughout the cytosol, and all contain starch and rubisco. During leaf development, the activities of phosphoenolpyruvate carboxylase (PEPC; which is cytosolic), rubisco, and pyruvate,Pi dikinase (PPDK) increase on a chlorophyll basis. As leaves mature, chloroplasts differentiate into two distinct structural and biochemical types that are spatially separated into the proximal and distal parts of the cell (the proximal end being closest to the center of the leaf). The early stages of this polarization are observed in intermediate leaves, and the polarization is fully developed in mature leaves. The chloroplasts in the distal ends of the cell have reduced grana and little starch, while those at the proximal ends have well-developed grana and abundant starch. In mature leaves, PPDK is expressed in chloroplasts at the distal end of the cells, while rubisco and adenosine diphosphate glucose (ADPG) pyrophosphorylase are selectively expressed in chloroplasts at the proximal end of the cell. Mitochondrial polarization also occurs during development as nicotinamide-adenine dinucleotide phosphate-malic enzyme (NAD-ME) and the photorespiratory enzyme glycine decarboxylase are expressed in mature but not young leaves and are localized in mitochondria at the proximal end of the cells. The data show that single-cell C(4) develops from a single pool of identical organelles that develop differential biochemical functions and spatial partitioning in the cell during maturation.     

Assembly of Functional Ribonucleoprotein Complexes by AU-rich Element RNA-binding Protein 1 (AUF1) Requires Base-dependent and -independent RNA Contacts

AU-rich element RNA-binding protein 1 (AUF1) regulates the stability and/or translational efficiency of diverse mRNA targets, including many encoding products controlling the cell cycle, apoptosis, and inflammation by associating with AU-rich elements residing in their 3′-untranslated regions. Previous biochemical studies showed that optimal AUF1 binding requires 33–34 nucleotides with a strong preference for U-rich RNA despite observations that few AUF1-associated cellular mRNAs contain such extended U-rich domains. Using the smallest AUF1 isoform (p37^AUF1) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37^AUF1 RNP complexes within a larger RNA context. In particular, p37^AUF1 binding to short AU-rich RNA targets was significantly stabilized by interactions with a 3′-purine residue and largely base-independent but non-ionic contacts 5′ of the AU-rich site. RNP stabilization by the upstream RNA domain was associated with an enhanced negative change in heat capacity consistent with conformational changes in protein and/or RNA components, and fluorescence resonance energy transfer-based assays demonstrated that these contacts were required for p37^AUF1 to remodel local RNA structure. Finally, reporter mRNAs containing minimal high affinity p37^AUF1 target sequences associated with AUF1 and were destabilized in a p37^AUF1-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs but also suggest how AUF1 binding could regulate protein and/or microRNA binding events at adjacent sites.     

Benzodiazepines and Tricyclic Antidepressant Plasma Levels

Twelve patients acted successfully as subjects to study what effect if any the benzodiazepines nitrazepam, diazepam, oxazepam, and chlordiazepoxide might have on steady-state plasma levels of nortriptyline and amitriptyline. No significant detectable effect was discovered. In view of the known interaction effects of other alternative tranquillizing drugs and hypnotics it seems reasonable to choose benzodiazepines wherever possible when anxiolytics or hypnotics need to be added during treatment of depression with tricyclic antidepressants.     

CITRATE AS THE PRECURSOR OF THE ACETYL MOIETY OF ACETYLCHOLINE

Abstract— Rat brain cortex slices were incubated with glucose labeled with either ³H or ¹⁴C in the 6-position. The ³H/¹⁴C ratios and the incorporation of radioactivity into lactate, citrate, malate and acetylcholine were determined. While the ³H/¹⁴C ratio of lactate was close to that of glucose, the ratios in the acetyl moiety of acetylcholine and the acetyl (C-4,5) portion of citrate decreased in a similar proportion. This was interpreted as indirect evidence for the participation of citrate as a precursor to the acetyl moiety of acetylcholine. Two inhibitors of the citrate cleavage pathway: n -butylmalonate, an inhibitor of citrate transport and (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase were studied for their effect on acetylcholine synthesis. N -butylmalonate (10 mM) and (-)-hydroxycitrate (7.5 mM) led to a decrease in the per cent of ¹⁴C recovered as acetylcholine. In each instance the ³H/¹⁴C ratio in acetylcholine was higher in the presence of inhibitor while the corresponding ratios in lactate and citrate (C-4.5) remained unchanged. From the results, it is suggested that citrate is involved in the transport mechanism of acetyl units from its site of synthesis in mitochondria to the site of acetylcholine synthesis in the cytosol.     

Interaction between embryogenic cultures of Scots pine and ectomycorrhizal fungi

 Embryogenic cell masses of three Scots pine (Pinus sylvestris) cell lines K779, K884 and K1009 were cultivated with the ectomycorrhizal (ECM) fungi Laccaria bicolor, L. proxima, Pisolithus tinctorius, Paxillus involutus and two strains of Suillus variegatus. The average growth ratio of the slowly proliferating cell line K1009 was improved by L. proxima and S. variegatus strain H, while of the rapidly proliferating lines K779 and K884 the non-mycorrhizal controls grew best. The fungi caused two distinct reactions in embryogenic cultures. In the positive reaction, the shape and light yellow colour of the cultures resembled the controls, while in the negative reaction the embryogenic cells became brown and necrotic and the fungi grew aggressively over them. These reactions to the fungi did not correlate completely with effects on the growth ratio. All the cell lines enhanced the radial growth of S. variegatus H and of P. tinctorius, while the Laccaria species and S. variegatus strain 1 thrived better alone. This study shows that early-stage embryogenic cells of Scots pine and ECM fungi are able to interact. As some fungi produced a positive reaction or even increased proliferation, they could be used to enhance somatic embryogenesis of Scots pine. Specific fungi might be used to induce the growth of slowly proliferating cell lines, and knowledge of positive cell line-fungus interactions could be useful in work with later stages of somatic embryogenesis, such as rooting. Accepted: 16 July 1998