Modest Interference with Actin Dynamics in Primary T Cell Activation by Antigen Presenting Cells Preferentially Affects Lamellal Signaling

Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC) signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation.     

Optimization of the medium for the yeast synthesis of biotin

In order to increase the yield of biotin produced by the culture Sporobolomyces pararoseus, the medium containing sucrose, asparagine, MgSO4 (NH4)2SO4, KH2PO4, vitamin complex and trace elements was optimized. With the aid of a fractional factor experiment (2(5-1)) and a complete factor experiment (2(4)), the proportion of constituents was chosen in such a way as to double biotin yield, i.e. to increase it to 55.25 micrograms/l. An enrichment of the medium with yeast autolysate, casein hydrolysate and peptone in the presence of adenine increased biotin yield to 105.7 micrograms/l and cell productivity from 6.1 to 8.0 micrograms/l dry biomass.     

Changes in Phenolics, Polyphenol Oxidase and its Isoenzyme Patterns in Relation to Resistance in Taro against Phytophthora colocasiae

The effect of Phytophthora leaf blight disease, caused by Phytophthora colocasiae Raciborski, on the accumulation of phenolics and polyphenol oxidase (PPO) activity in ex vitro plants was studied in three resistant (DP‐25, Duradim and Jhankri) and one susceptible (N‐118) genotypes of taro [Colocasia esculenta (L). Schott]. The inoculation of taro leaves with P. colocasiae spores resulted in a quantitative change in both biochemical parameters and induction of PPO isoforms in resistant genotypes. The amount of phenolics was increased owing to blight by 68.02%, 58.87%, 52.67% and 11.50% in DP‐25, Duradim, Jhankri and N‐118, respectively. The per cent increase in PPO under stress over non‐stress condition was also highest in DP‐25 (49.14%) followed by Duradim (41.56%), Jhankri (40.55%) and N‐118 (17.08%). The resistant genotypes showed higher activity of PPO as compared with susceptible ones, which was reflected through its banding pattern in isoenzyme analysis, detecting four different isoforms. The intensity of the bands was higher in the resistant genotypes than in susceptible N‐118. The appearance of high intensity bands and/or reduction in the intensity of particular isoform(s) in the zymograms of all the three resistant taro genotypes studied, led to the apparent conclusion of linking PPO isoenzyme expression with blight resistance in taro. The blight incidence (per cent leaf infection and leaf area infection) was lower in the resistant genotypes than in susceptible, N‐118. The yield reduction owing to blight was below 20% in DP‐25, Jhankri and Duradim, while the same was more than 40% in N‐118. The phenolics and PPO activity was negatively correlated with disease incidence and yield reduction owing to blight. Based on the results of disease incidence, biochemical contents and yield, the pattern of stress tolerance was DP‐25 > Duradim > Jhankri > N‐118. The studied parameters, i.e. phenolics and PPO could be used as biochemical markers for leaf blight stress tolerance studies in taro.     

Errors in egg-laying by female Common Cuckoo Cuculus canorus in nests of its common host

Dozens of studies have documented that brood parasites are well adapted to a brood parasitic lifestyle but not all parasitism events are successful. Co-evolution between brood parasites and their hosts is a dynamic process so it is reasonable to expect that a female brood parasite may commit errors during egg deposition by laying her eggs outside the laying period of the host, with consequent impacts on her fitness. Using an extensive dataset from a long-term study, we evaluated egg-laying patterns and errors related to the timing of egg-laying in the Common Cuckoo Cuculus canorus (hereafter ‘Cuckoo’). Specifically, we tested whether the Cuckoo avoids laying before or on the day of host clutch initiation to reduce the risk of rejection of parasitic eggs, whether laying errors will be more frequent in periods with a lack of active host nests, and whether the laying errors will be more frequent in periods with intense Cuckoo parasitism and a consequent lack of suitable host nests. We found that about one-third of Cuckoo eggs were laid on the host clutch initiation day or 1 day before, and the percentage of Cuckoo eggs laid decreased thereafter. Surprisingly, the probability of Cuckoo egg acceptance by the hosts was not affected by the egg-laying stage of the host clutch. Errors in the timing of egg-laying with fatal consequences (i.e. those precluding Cuckoo hatching because of laying in incubated or deserted clutches) were recorded in about 5% of cases. Only laying date of a Cuckoo egg had a significant effect on the probability of errors, which increased during the breeding season. This may be related to the higher number of deserted and incubated host nests at the site at the end of the breeding season. Errors in egg-laying may be attributed to young and inexperienced females but also impaired body condition or intraspecific competition may cause this behaviour. Future studies, which will test these possible explanations, will help to understand better the mechanism of co-evolutionary arms races and differences between host specialist and generalist brood parasites in various host–parasite systems.     

Comparative mapping of loci controlling winter survival and related traits in oilseed Brassica rapa and B. napus

Winter survival is an important characteristic of oilseedBrassica that is seeded in the fall in northern climates,and it may be affected by genetic variation for other cold-regulated traits,such as freezing tolerance and vernalization responsive flowering time. Weanalyzed immortalized populations of oilseed Brassica rapa(recombinant inbred lines) and B. napus (double haploidlines) derived from crosses of annual and biennial types in order to comparethe map positions and effects of quantitative trait loci controlling wintersurvival, nonacclimated and acclimated freezing tolerances, and flowering time.The B. napus population was evaluated in multiple winters,and six of the 16 total significant QTL for winter survival were detected inmore than one winter. Correspondence in the map positions of QTL controllingdifferent traits within species provided evidence that some alleles causinggreater acclimated freezing tolerance and later flowering time also contributedto increased winter survival. Correspondence in the map positions of QTLbetween species provided evidence for allelic variation at homologous loci inB. rapa and B. napus. The potentialrole of some candidate genes in regulating these traits is discussed.     

Reversible change in thiol redox status of the insulin receptor alpha-subunit in intact cells

In this study, we used maleimidobutyrylbiocytin to examine possible alteration that may occur in the redox state of the insulin receptor (IR) sulfhydryl groups in response to reduced glutathione (GSH) or N-acetyl-L-cysteine (NAC). Short-term treatment of intact cells expressing large numbers of IR with GSH or NAC led to a rapid and reversible reduction of IR alpha-subunit disulfides, without affecting the receptor beta-subunit thiol reactivity. The overall integrity of the oligomeric structure of IR was maintained, indicating that neither class I nor class II disulfides were targeted by these agents. Similar findings were obtained in cells transfected with IR mutants lacking cysteine524, one of the class I disulfides that link the two IR alpha-subunits. Membrane-associated thiols did not participate in GSH- or NAC-mediated reduction of IR alpha-subunit disulfides. No difference in insulin binding was observed in GSH-treated cells; however, ligand-mediated increases in IR autophosphorylation, tyrosine phosphorylation of cellular substrates, and dual phosphorylation of the downstream target mitogen-activated protein kinase were inhibited at concentrations of GSH (10 mM or greater) that yielded a significant increase in IR alpha-subunit thiol reactivity. GSH did not affect IR signaling in the absence of insulin. Our results provide the first evidence that the IR alpha-subunit contains a select group of disulfides whose redox status can be rapidly altered by the reducing agents GSH and NAC.     

Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake.

A novel, positive read-out assay that quantifies only sequence-specific nuclear activity of antisense oligonucleotides was used to evaluate morpholino and 2'-O-methyl sugar-phosphate oligonucleotides. The assay is based on modification of the splicing pathway of human beta-globin pre-mRNA. In addition, scrape-loading of cells with oligonucleotides allows the separate assessment of intracellular antisense activity of the oligonucleotides and their ability to penetrate the cell membrane barrier. The results show that, with scrape-loading, the morpholino oligonucleotides were approximately 3-fold more effective in their intrinsic antisense activity than alternating phosphodiester/phosphorothioate 2'-O-methyl-oligoribonucleotides and 6-9- and almost 200-fold more effective than the exclusively phosphorothioate and phosphodiester derivatives, respectively. The morpholino oligonucleotides were over 20-fold more effective than the phosphorothioate 2'-O-methyl-oligoribonucleotides in free uptake from the culture media. The antisense activity of the morpholino oligonucleotides was detectable not only in monolayer HeLa cells but also in suspension K562 cells. Time course experiments suggest that both the free uptake and efflux of morpholino oligonucleotides are slow.