Inside Cover: Generalized spectral light scatter models of diverse bacterial colony morphologies (J. Biophotonics 12/2019)

The forward elastic‐light‐scattering pattern of a bacterial colony reflects its morphological characteristics. Three bacteria genera whose colonies having convex, crateriform, or irregular elevation were investigated to study the correlation between the morphology and the scattering pattern of the colony. The difference in the colony elevation produced distinct shapes of light diffraction in the scattering pattern, resulting circular diffraction rings or scattered light. Further details can be found in the article by Iyll‐Joon Doh, Jennifer Sturgis, Diana V. Sarria Zuniga, et al. ( e201900149 ).

     

Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus

The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (-log EC50 shift = -1.49 +/- 0.16 and E(max) inhibition for 100 microM ACh = 30%) more than the same levels of histamine contraction. The M2 receptor-selective antagonist methoctramine (1 microM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric acid (30 nM), demonstrated by abolition of contractile and inositol phosphate responses to ACh, muscarinic recontractile responses were obtained in U-46619-precontracted tissues fully relaxed with isoproterenol. Methoctramine antagonized recontraction, with pK(B) (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In M3-inactivated tissues, methoctramine augmented the isoproterenol relaxant potency in U-46619-contracted bronchus and reversed the ACh-induced inhibition of isoproterenol cAMP accumulation. These results indicate that M2 receptors cause indirect contraction of human bronchus by reversing sympathetically mediated relaxation and contribute to cholinergic functional antagonism.     

Genetic linkage map of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) and localization of a major QTL for powdery mildew resistance

Powdery mildew caused by Podosphaera xanthii has become a major problem in melon since it occurs all year round irrespective of the growing system. The TGR-1551 melon genotype was found to be resistant to several melon diseases, among them powdery mildew. However, the corresponding resistance genes have been never mapped. We constructed an integrated genetic linkage map using an F2 population derived from a cross between the multi-resistant genotype TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro’. The map spans 1,284.9 cM, with an average distance of 3.6 cM among markers, and consists of 354 loci (188 AFLP, 39 RAPD, 111 SSR, 14 SCAR/CAPS/dCAPS, and two phenotypic traits) distributed in 14 linkage groups. QTL analysis identified one major QTL (Pm-R) on LG V for resistance to races 1, 2, and 5 of powdery mildew. The PM4-CAPS marker is closely linked to the Pm-R QTL at a genetic distance of 1.9 cM, and the PM3-CAPS marker is located within the support interval of this QTL. These codominant markers, together with the map information reported here, could be used for melon breeding, and particularly for genotyping selection of resistance to powdery mildew in this vegetable crop species.     

A functional role for vimentin intermediate filaments in the metabolism of lipoprotein-derived cholesterol in human SW-13 cells.

Numerous studies have indicated that cytoplasmic intermediate filaments (cIFs) can associate with cellular lipids. To determine if these interactions might have functional consequences, we have studied the lipid metabolism of human SW-13 adrenal tumor cell lines that either contain vimentin-type cIFs (vim+) or lack any detectable cIF network (vim-). Although there were no significant differences in phospholipid or glyceride synthesis, vim- cell lines had elevated levels of cholesterol synthesis and decreased cholesterol esterification, compared with vim+ cells. These differences in cholesterol synthesis and esterification were found to be due to an impaired ability of vim- cells to utilize low density lipoprotein (LDL)-derived cholesterol, although receptor-mediated endocytosis of LDL and the capacity of these cells to esterify endogenously produced cholesterol were not affected. Expression of a mouse vimentin cDNA in stably transfected cell lines, derived from vim- cells, restored the capacity of these cells to utilize LDL cholesterol. The uptake and metabolism of [3H]cholesterol linoleate-loaded LDL showed that the impaired ability of vim- cells to esterify LDL cholesterol was not associated with an accumulation of cellular free cholesterol but rather an increase in the appearance of [3H]cholesterol in the culture medium. These studies indicate that in SW-13 cells, the intracellular movement of LDL-derived cholesterol from the lysosome to the site of esterification is a vimentin-dependent process.     

Proteolytic activity assayed by subcellular localization switching of a substrate

An approach to assay proteolytic activity in vivo by altering the subcellular localization of a labelled substrate was demonstrated. The assay included a protein shuttling between different cellular compartments and a site-specific recombinant protease. The shuttle protein used was the human immunodeficiency virus type 1 (HIV-1) Rev protein tandemly fused to the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), while the protease was the site-specific protease VP24 from the herpes simplex virus type 1 (HSV-1). The fluorescent proteins in the Rev fusion protein were separated by a cleavage site specific for the VP24 protease. When co-expressed in COS-7 cells proteolysis was observed by fluorescence microscopy as a shift from a predominantly cytoplasmic localization of the fusion protein RevEGFP to a nuclear localization while the RFP part of the fusion protein remained in the cytoplasm. The cleavage of the fusion protein by VP24 was confirmed by Western blot analysis. The activity of VP24, when tagged N-terminally by the Myc-epitope, was found to be comparable to VP24. These results demonstrates that the activity and localization of a recombinantly expressed protease can be assessed by protease-mediated cleavage of fusion proteins containing a specific protease cleavage site.     

Role of leaf glandular trichomes of melon plants in deterrence of Aphis gossypii Glover

External characteristics of the leaf epidermis and their effects on behaviour of Aphis gossypii Glover were evaluated in two Cucumis melo L. genotypes, ‘Bola de Oro’ (aphid susceptible) and TGR‐1551 (aphid resistant) in order to explore their role in the early rejection of TGR‐1551 by this aphid. No differential effects of epicuticular waxes on aphid behaviour were observed. The type, distribution and number of trichomes on melon leaves were also studied. Pubescence in melon, measured as the number of non‐glandular trichomes per cm², was not sufficient to prevent aphid settling. However, there was a high density of type I glandular trichomes on leaves of the aphid‐resistant genotype. According to microscopic observations and stain testing, these trichomes store and secrete phenols and flavonoids. Free‐choice tests were conducted to determine the effect of these glandular trichomes on A. gossypii preference, revealing that aphids reject leaf disks of TGR‐1551 from the onset of the experiment. Additional experiments after removal of leaf type I glandular trichome exudates showed that A. gossypii preferred washed TGR‐1551 leaf disks over unwashed disks, while this effect was not observed in experiments using washed and unwashed ‘Bola de Oro’ leaf disks. These results suggest that a high density of glandular trichomes and chemicals secreted by them deter A. gossypii and disturb aphid settling on TGR‐1551.     

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2

Trafficking of AMPA-type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1-binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR-mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21-GRIP1 binding is crucial for GluR2-AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.