Fusogenic Pairings of Vesicle-Associated Membrane Proteins (VAMPs) and Plasma Membrane t-SNAREs – VAMP5 as the Exception

Background: Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities. Methodology/Principal Findings: In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of b-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/ SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30–50 % lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction. Conclusions/Significance: These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs

Frequency and spectrum of M2 mutants and genetic variability in cyto-agronomic characteristics of fenugreek induced by caffeine and sodium azide

Trigonella foenum graecum L. (Fenugreek) is a valuable medicinal plant cultivated for decades for its therapeutic characteristics. Still no pronounced improvement concerning wild form was accomplished as it is a self-pollinating crop. Induced mutagenesis is encouraged as a remarkable tool on this plant to circumvent the genetic bottleneck of cultivated germplasms. As a result, novel allelomorphic combinations for short-term agronomic attributes were developed. Fenugreek cultivar Pusa Early Bunching, selected for the present experiment, was mutagenized with five doses (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%) of caffeine and sodium azide (SA) to evaluate its impact on the qualitative and quantitative traits of M1 and M2 generation conducted in a Complete Randomized Block Design (CRBD), replicated five times during 2019–2020 and 2020–2021, respectively. The frequency of induced phenotypic variations was assessed in M2 progenies, resulting in the identification and isolation of a broad spectrum of mutants with altered phenotypes. Mutagenic effectiveness and efficiency were found to be maximum at lower concentrations of the mutagen treatments and highest in SA, followed by caffeine. Various morphological mutants with modified characters were observed at different concentrations in M2 generation. The spectrum of mutations was wider in SA than in caffeine, as caffeine produced 51 while SA produced 54 individual mutants under seven major categories. The maximum frequency of morphological mutants was associated with leaf, followed by plant size, plant growth habit, pod, seed size, seed shape, and seed color. Morphological and structural variations in the guard cells of stomata and seeds were observed through scanning electron microscopy. The variations created in the economically important traits may enrich the genetic diversity of this plant species. Moreover, these morphological mutants may serve as a source of elite genes in further breeding programs of fenugreek

LRIT3 expression in cone photoreceptors restores post-synaptic bipolar cell signalplex assembly and partial function in Lrit3−/− mice

Summary: Complete congenital stationary night blindness (cCSNB) is a heterogeneous disorder characterized by poor dim-light vision, myopia, and nystagmus that is caused by mutations in genes critical for signal transmission between photoreceptors and depolarizing bipolar cells (DBCs). One such gene, LRIT3, is required for assembly of the post-synaptic signaling complex (signalplex) at the dendritic tips of DBCs, although the number of signalplex components impacted is greater in cone DBCs (all components) than in rod bipolar cells (only TRPM1 and Nyctalopin). Here we show that rAAV-mediated expression of LRIT3 in cones results in robust rescue of cone DBC signalplex components and partially restores downstream visual function, as measured by the light-adapted electroretinogram (ERG) b-wave and electrophysiological recordings of bipolar cells (BCs) and RGCs. These data show that LRIT3 successfully restores partial function to cone DBCs most likely in a trans-synaptic manner, potentially paving the way for therapeutic intervention in LRIT3-associated cCSNB

Recent advancements in molecular marker-assisted selection and applications in plant breeding programmes

Abstract Background DNA markers improved the productivity and accuracy of classical plant breeding by means of marker-assisted selection (MAS). The enormous number of quantitative trait loci (QTLs) mapping read for different plant species have given a plenitude of molecular marker-gene associations. Main body of the abstract In this review, we have discussed the positive aspects of molecular marker-assisted selection and its precise applications in plant breeding programmes. Molecular marker-assisted selection has considerably shortened the time for new crop varieties to be brought to the market. To explore the information about DNA markers, many reviews have been published in the last few decades; all these reviews were intended by plant breeders to obtain information on molecular genetics. In this review, we intended to be a synopsis of recent developments of DNA markers and their application in plant breeding programmes and devoted to early breeders with little or no knowledge about the DNA markers. The progress made in molecular plant breeding, plant genetics, genomics selection, and editing of genome contributed to the comprehensive understanding of DNA markers and provides several proofs on the genetic diversity available in crop plants and greatly complemented plant breeding devices. Short conclusion MAS has revolutionized the process of plant breeding with acceleration and accuracy, which is continuously empowering plant breeders around the world

Allicin from garlic suppresses TNF-α and augments IFN-γ expressions in monocyte cultures from patients with Vaginitis

Objective: We tried to explore beneficial effects of allicin- a natural antioxidant from garlic in the possible management of vaginitis. 
Methods: Peripheral blood mononuclear cells (PBMC’s) were isolated from blood of patients having vaginal infections as well as normal healthy subjects as per our published protocol. Monocytes (MN’s) from above PBMC’s were adhered, rested overnight and cultured without or with varying doses of allicin (0-500 ng/ml). Some cultures received SN50 and SN50M (100 ug/ml). After 24 hrs cultures, the cells were harvested and the supernatants subjected to secreted TNF-α and sIFN-γ assays by ELISA. Treated/untreated harvested cells were subjected to glutathione peroxidase (GPx) activity assay.
Results: We show involvement of inflammatory cytokines and reactive oxygen species (ROS) in vaginitis. Augmented secreted TNF-α and decreased sIFN-γ levels were observed in monocyte cultures of vaginal patients. Relative to healthy controls, a decrease by 2.5-fold in glutathione peroxidase (GPx) activity in cultures of vaginal patients was observed. Treatment with allicin from garlic appreciably decreased secreted TNF-α with simultaneous amelioration in sIFN-γ and GPx activity. Allicin (500 ng/ml) ameliorated GPx activity by ~ 2.16-folds in patient monocyte cultures. The anti-oxidant and anti-inflammatory actions of allicin induced in monocyte cultures of vaginal infection patients were mediated via NF-κB.
Conclusion: Thus, the above results may help in understanding the use of allicin as an adjunct in vaginal infection therapy, and in turn, may address the globally female health.
&#xa

A missense mutation in Grm6 reduces but does not eliminate mGluR6 expression or rod depolarizing bipolar cell function.

GRM6 encodes the metabotropic glutamate receptor 6 (mGluR6) used by retinal depolarizing bipolar cells (DBCs). Mutations in GRM6 lead to DBC dysfunction and underlie the human condition autosomal recessive complete congenital stationary night blindness. Mouse mutants for Grm6 are important models for this condition. Here we report a new Grm6 mutant, identified in an electroretinogram (ERG) screen of mice maintained at The Jackson Laboratory. The Grm6(nob8) mouse has a reduced-amplitude b-wave component of the ERG, which reflects light-evoked DBC activity. Sequencing identified a missense mutation that converts a highly conserved methionine within the ligand binding domain to leucine (p.Met66Leu). Consistent with prior studies of Grm6 mutant mice, the laminar size and structure in the Grm6(nob8) retina were comparable to control. The Grm6(nob8) phenotype is distinguished from other Grm6 mutants that carry a null allele by a reduced but not absent ERG b-wave, decreased but present expression of mGluR6 at DBC dendritic tips, and mislocalization of mGluR6 to DBC somas. Consistent with a reduced but not absent b-wave, there were a subset of retinal ganglion cells whose responses to light onset have times to peak within the range of those in control retinas. These data indicate that the p.Met66Leu mutant mGluR6 is trafficked less than control. However, the mGluR6 that is localized to the DBC dendritic tips is able to initiate DBC signal transduction. The Grm6(nob8) mouse extends the Grm6 allelic series and will be useful for elucidating the role of mGluR6 in DBC signal transduction and in human disease.NEW & NOTEWORTHY This article describes a mouse model of the human disease complete congenital stationary night blindness in which the mutation reduces but does not eliminate GRM6 expression and bipolar cell function, a distinct phenotype from that seen in other Grm6 mouse models. J Neurophysiol 2017 Aug; 118(2):845-854