A unique pollen wall mutation in the family Compositae: ultrastructure and genetics

During a routine screening of pollen fertility in the n = 2 chromosome race of Haplopappus gracilis, a spineless pollen wall mutation was discovered that renders the otherwise functional pollen grains completely unrecognizable as Compositae pollen. Normal Haplopappus pollen is characterized by an outer layer, the ektexine, consisting of large spines supported by a roof (tectum), which in turn is supported by collumellae that are joined basally. A large cavity (cavea) stretches from aperture to aperture and separates columellae bases from the final ektexine unit, the foot layer. The spines, tectum, columellae, and columellae bases are filled with perforations (internal foramina), while the foot layer is without them. Immediately underlying the foot layer is a thickened, lamellate, disrupted, internal foramina-free second exine layer, the endexine. In contrast, the mutant pollen ektexine is a jumble of components with randomly dispersed spines as the only clearly definable unit. The endexine layer is similar to the endexine in normal pollen. The mutation apparently disrupts only the organization of ektexine units, and mutant pollen appears to be without the caveae and foot layer characteristic of normal pollen. In genetic tests, the mutant allele is recessive. There is a simple Mendelian pattern of inheritance of the mutant gene, and its phenotype is under sporophytic control.     

Role of AWD/Nucleoside Diphosphate Kinase in Drosophila Development

The abnormal wing discs gene of Drosophila encodes a soluble protein with nucleosidediphosphate kinase activity. This enzymic activity is necessary for the biological function ofthe abnormal wing discs gene product. Complete loss of function, i.e., null, mutations causelethality after the larval stage. Most larval organs in such null mutant larvae appear to benormal, but the imaginal discs are small and incapable of normal differentiation.Killer-of-prune is a neomorphic mutation in the abnormal wing discs gene. It causes dominant lethalityin larvae that lack prune gene activity. The Killer-of-prune mutant protein may have alteredsubstrate specificity. Null mutant larvae have a low level of nucleoside diphosphate kinaseactivity. This suggests that there may be additional Drosophila genes that encode proteinswith nucleoside dipthosphate kinase activity. Candidate genes have been found in theDrosophila genome.     

Genetic Complexity in a Drosophila Model of Diabetes-Associated Misfolded Human Proinsulin

Drosophila melanogaster has been widely used as a model of human Mendelian disease, but its value in modeling complex disease has received little attention. Fly models of complex disease would enable high-resolution mapping of disease-modifying loci and the identification of novel targets for therapeutic intervention. Here, we describe a fly model of permanent neonatal diabetes mellitus and explore the complexity of this model. The approach involves the transgenic expression of a misfolded mutant of human preproinsulin, hINS^C96Y, which is a cause of permanent neonatal diabetes. When expressed in fly imaginal discs, hINS^C96Y causes a reduction of adult structures, including the eye, wing, and notum. Eye imaginal discs exhibit defects in both the structure and the arrangement of ommatidia. In the wing, expression of hINS^C96Y leads to ectopic expression of veins and mechano-sensory organs, indicating disruption of wild-type signaling processes regulating cell fates. These readily measurable “disease” phenotypes are sensitive to temperature, gene dose, and sex. Mutant (but not wild-type) proinsulin expression in the eye imaginal disc induces IRE1-mediated XBP1 alternative splicing, a signal for endoplasmic reticulum stress response activation, and produces global change in gene expression. Mutant hINS transgene tester strains, when crossed to stocks from the Drosophila Genetic Reference Panel, produce F₁ adults with a continuous range of disease phenotypes and large broad-sense heritability. Surprisingly, the severity of mutant hINS-induced disease in the eye is not correlated with that in the notum in these crosses, nor with eye reduction phenotypes caused by the expression of two dominant eye mutants acting in two different eye development pathways, Drop (Dr) or Lobe (L), when crossed into the same genetic backgrounds. The tissue specificity of genetic variability for mutant hINS-induced disease has, therefore, its own distinct signature. The genetic dominance of disease-specific phenotypic variability in our model of misfolded human proinsulin makes this approach amenable to genome-wide association study in a simple F₁ screen of natural variation.     

Increasing stability of antibody via antibody engineering: Stability engineering on an anti‐hVEGF

Antibody stability is very important for expression, activity, specificity, and storage. This knowledge of antibody structure has made it possible for a computer‐aided molecule design to be used to optimize and increase antibody stability. Many computational methods have been built based on knowledge or structure, however, a good integrated engineering system has yet to be developed that combines these methods. In the current study, we designed an integrated computer‐aided engineering protocol, which included several successful methods. Mutants were designed considering factors that affected stability and multiwall filter screening was used to improve the design accuracy. Using this protocol, the thermo‐stability of an anti‐hVEGF antibody was significantly improved. Nearly 40% of the single‐point mutants proved to be more stable than the parent antibody and most of the mutations could be stacked effectively. The T₅₀ also improved about 7°C by combinational mutation of seven sites in the light chain and three sites in the heavy chain. Data indicate that the protocol is an effective method for optimization of antibody structure, especially for improving thermo‐stability. This protocol could also be used to enhance the stability of other antibodies. Proteins 2014; 82:2620–2630. © 2014 Wiley Periodicals, Inc.     

Auxin conjugated to fluorescent dyes – a tool for the analysis of auxin transport pathways

Auxin is a small molecule involved in most processes related to plant growth and development. Its effect usually depends on the distribution in tissues and the formation of concentration gradients. Until now there has been no tool for the direct tracking of auxin transport at the cellular and tissue level; therefore the majority of studies have been based on various indirect methods. However, due to their various restrictions, relatively little is known about the relationship between various pathways of auxin transport and specific developmental processes. We present a new research tool: fluorescently labelled auxin in the form of a conjugate with two different fluorescent tracers, FITC and RITC, which allows direct observation of auxin transport in plant tissues. Chemical analysis and biological tests have shown that our conjugates have auxin‐like biological activity and transport; therefore they can be used in all experimental systems as an alternative to IAA. In addition, the conjugates are a universal tool that can be applied in studies of all plant groups and species. The conjugation procedure presented in this paper can be adapted to other fluorescent dyes, which are constantly being improved. In our opinion, the conjugates greatly expand the possibilities of research concerning the role of auxin and its transport in different developmental processes in plants.     

Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis

Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in the absence of ATP-analogue inhibitors. This has limited the use of the strain for use in these settings. Here, we used in vivo selection for intragenic suppressor mutations of cdc2-as that restore full function in the absence of ATP-analogues. The cdc2-asM17 underwent meiosis and produced viable spores to a similar degree to the wild-type strain. The suppressor mutation also rescued the sensitivity of the cdc2-as strain to high and low temperature, genotoxins and an anti-microtubule drug. We have used cdc2-asM17 to show that Cdc2 activity is required to maintain the activity of the spindle assembly checkpoint. Furthermore, we also demonstrate that maintenance of the Shugoshin Sgo1 at meiotic centromeres does not require Cdc2 activity, whereas localization of the kinase aurora does. The modified cdc2-asM17 allele can be thus used to analyse many aspects of cell-cycle-related events in fission yeast.     

Expression of biopterin transporter (BT1) protein in Leishmania

The present work focuses on the growth phase regulated expression of biopterin transporter gene (BT1) from the LD1 locus on chromosome 35 of Leishmania donovani. Antiserum against recombinant BT1 detected a polypeptide of 45 kDa of equal intensity at lag, log and stationary phases of promastigote growth, both in L. donovani strain LSB-7.1 (MHOM/BL/67/ITMAP263), and strain LSB-146.1 (HOM/IR/95/X81), a natural isolate from Isfehan, Iran that caused cutaneous leishmaniasis. However, in both these strains an additional polypeptide of higher molecular mass (50 kDa) was also observed during lag phase only. In addition, polypeptides of 40, 20, 18 and 16 kDa were seen only during the lag and log phases of both strains. Analysis of L. donovani single, double and triple (null) BT1 knockout mutants confirmed that the 45-kDa polypeptide was the BT1 gene product, as it was absent in the null mutant. These results indicate that 45-kDa BT1 protein in Leishmania is consistently and constitutively expressed in all the growth stages of the parasite.     

IL-1beta promotes neurite outgrowth by deactivating RhoA via p38 MAPK pathway

Expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1β) is increased following the nervous system injury. Generally IL-1β induces inflammation, leading to neural degeneration, while several neuropoietic effects have also been reported. Although neurite outgrowth is an important step in nerve regeneration, whether IL-1β takes advantages on it is unclear. Now we examine how it affects neurite outgrowth. Following sciatic nerve injury, expression of IL-1β is increased in Schwann cells around the site of injury, peaking 1 day after injury. In dorsal root ganglion (DRG) neurons and cerebellar granule neurons (CGNs), neurite outgrowth is inhibited by the addition of myelin-associated glycoprotein (MAG), activating RhoA. IL-1β overcomes MAG-induced neurite outgrowth inhibition, by deactivating RhoA. Intracellular signaling experiments reveal that p38 MAPK, and not nuclear factor-kappa B (NF-κB), mediated this effect. These findings suggest that IL-1β may contribute to nerve regeneration by promoting neurite outgrowth following nerve injury.