Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.

Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.     

Starch as a sugar reservoir for nematode-induced syncytia

The plant parasitic nematode Heterodera schachtii invades the roots of Arabidopsis thaliana to induce nematode feeding structures in the central cylinder. During nematode development, the parasites feed exclusively from these structures. Thus, high sugar import and specific sugar processing of the affected plant cells is crucial for nematode development. In the present work, we found starch accumulation in nematode feeding structures and therefore studied the expression genes involved in the starch metabolic pathway. The importance of starch synthesis was further shown using the Atss1 mutant line. As it is rather surprising to find starch accumulation in cells characterised by a high nutrient loss, we speculate that starch serves as long- and short-term carbohydrate storage to compensate the staggering feeding behaviour of the parasites.Key words: Heterodera schachtii, Arabidopsis, nematode, starch metabolism, syncytiaThe obligate plant parasitic nematode Heterodera schachtii is entirely dependent on a system of nutrient supply provided by the plant. Host plants—among those the model plant Arabidopsis thaliana—have to endure invasion of second stage juveniles and the establishment of nematode feeding structures in the plant''s vascular cylinder. For induction of the specific feeding structures, the juveniles pierce one single plant cell with their stylet and inject secretions, thus triggering the formation of a syncytium by local cell walls dissolutions.¹ Further, the central vacuole of the syncytial cells disintegrates, nuclei enlarge and many organelles proliferate.¹ About 24 hours after feeding site induction, the nematode juveniles start feeding in repetitive cycles.² Syncytia have previously been described as strong sinks in the plant''s transport system.³ Thus, in the recent years several studies were carried out to discover solute supply to syncytial cells.^4–7 To our present knowledge, syncytia are symplasmically isolated in the first days of nematode development. During that period, the nematodes depend on transport protein activity in the syncytia plasmamembranes. At later stages plasmodesmata appear to open to the phloem elements, facilitating symplasmic transport.Incoming solutes may either be taken up by the feeding nematode or are synthesised and catalysed by the syncytium''s metabolism. Due to the microscopically observable high density of the cytosol¹ and the increased osmotic pressure,⁸ syncytia appear to accumulate high solute concentrations. In fact, significantly increased sucrose levels have been found in syncytia in comparison to non-infected control roots.⁷ In case of high sugar levels, plant cells generally synthesize starch in order to reduce emerging osmotic stress.⁹ The aim of the work of Hofmann et al.,¹⁰ was to elucidate if starch is utilised as carbohydrate storage in nematode-induced syncytia and to study expression of genes involved in starch metabolism with an emphasis on nematode development.Starch levels of nematode induced syncytia and roots of non-infected plants grown on sand/soil culture were measured by high performance liquid chromatography (HPLC). The results showed a high accumulation of starch in syncytia that was steadily decreasing during nematode development. The accumulation of starch could further be localised within syncytial cells by electron microscopy. Based on these results, we studied the gene expression of the starch metabolic pathway by Affymetrix gene chip analysis. About half of the 56 involved genes were significantly upregulated in syncytia compared to the control and only two genes were significantly downregulated. Thus, the high induction of the gene expression is consistent with the high starch accumulation. Finally, we applied an Arabidopsis mutant line lacking starch synthase I expression that has been described previously.¹¹ Starch synthase I was the second highest upregulated gene in syncytia. It catalyses the linkage of ADP-glucose to the non-reducing end of an a-glucan, forming the linear glucose chains of amylopectin. In a nematode infection assay we were able to prove the significant importance of the gene for nematode development.With the presented results, we can unambiguously prove the accumulation of starch and the induction of the gene expression of the starch metabolic pathway in nematode-induced syncytia. The primary question however is: why do syncytia accumulate soluble sugars and starch although their metabolism is highly induced and nematodes withdraw solutes during continuously repeating feeding cycles?One explanation may be found where least expected—in nematode feeding. It is the feeding activity that induced solute import mechanisms into syncytia resulting in a newly formed sink tissue. However, during moulting events to the third, the fourth juvenile stage and to the adult stage nematodes interrupt feeding for about 20 hours.² During this period sugar supply mechanisms will most probably not be altered thus leading to increasing levels of sugars in the syncytium. Starch may serve as short-term carbohydrate buffering sugar excess. Further, starch may serve as long-term carbohydrate storage during nematode development. In the early stages of juvenile development nematodes withdraw considerably small quantities (about 0,8-times the syncytium volume a day).¹² At later stages, nutrient demand increases so that adult fertilised females require 4-times the syncytium volume per day in order to accomplish egg production.¹² Thus, excessive sugar supply in the first days may be accumulated as starch that gets degraded at later stages when more energy is required from the parasites. Consequently, starch reserve serves as both short-term and long-term carbohydrate storage in nematode-induced syncytia in order to buffer changing feeding pattern of the parasites.? Open in a separate windowFigure 1Arabidopsis wild-type Columbia-0 plants were grown in sand/soil culture. Nematode-induced syncytia and non-infected control roots were harvested at 10, 15 and 20 days after inoculation (dai) and starch content was measured as glucose (Glc) equivalents. Values are means ± SE, n = 3. Different letters indicate significant variations (p < 0.05). © ASPBOpen in a separate windowFigure 2Transmission electron microscope picture of a cross-section of a syncytium associated with female fourth stage juvenile (H. schachtii) induced in roots of Arabidopsis. Bar = 2 µm. S, syncytium; Se, sieve tube; arrow, plastid; asterisk, starch granule. © ASPB     

Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study

Introduction  

Improvement of rheumatoid arthritis (RA) during pregnancy has been causatively associated with increased galactosylation of immunoglobulin G (IgG) N-glycans. Since previous studies were small, did not include the postpartum flare and did not study sialylation, these issues were addressed in the present study.     

Statistical Issues in Modeling Chronic Disease in Cohort Studies

Observational cohort studies of individuals with chronic disease provide information on rates of disease progression, the effect of fixed and time-varying risk factors, and the extent of heterogeneity in the course of disease. Analysis of this information is often facilitated by the use of multistate models with intensity functions governing transition between disease states. We discuss modeling and analysis issues for such models when individuals are observed intermittently. Frameworks for dealing with heterogeneity and measurement error are discussed including random effect models, finite mixture models, and hidden Markov models. Cohorts are often defined by convenience and ways of addressing outcome-dependent sampling or observation of individuals are also discussed. Data on progression of joint damage in psoriatic arthritis and retinopathy in diabetes are analysed to illustrate these issues and related methodology.     

Amino acids in carbonaceous chondrites

For almost 20 years laboratory experiments have advanced the concepts of chemical evolution, particularly with regard to formation of the amino acids. What has been generally lacking is concrete natural evidence for this chemical evolution hypothesis. The recent development of sophisticated analytical techniques and availability of carbonaceous chondrites with a minimum of terrestrial contamination has resulted in the identification of amino acids which provide strong evidence for a natural extraterrestrial chemical synthesis. Since the initial find in the Murchison meteorite (a type II carbonaceous chondrite) of both protein and nonprotein amino acids and amino acids with nearly equal abundances of D and L isomers, further studies have been carried out. These studies have revealed the presence of at least 35 amino acids; the population consists of a wide variety of linear, cyclic and polyfunctional amino acids which shows a trend of decreasing concentration with increasing carbon number. Investigations of the Murray meteorite (a type II carbonaceous chondrite) has produced similar results, but studies of the Orgueil meteorite (a type I carbonaceous chondrite) show only a limited suite of amino acids, some of which appear to be indigenous while others appear to be terrestrial contaminants. A sample of the Murchison meteorite was extracted with D₂O and in addition to free amino acids, showing no deuterium incorporation, some amino acids showed the presence of deuterium suggesting either a precursor(s) or hydrogendeuterium exchange which require(s) formation of carbon-hydrogen bonds.