Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.     

A rapid and efficient method for the extraction of total DNA from the sweet potato and its related species

Secondary metabolites, latex/mucilagenous secretions, polysaccharides, and proteins interfere with the extraction of high-quality, restrictable total cellular DNA from sweet potato [Ipomoea batatas (L.) Lamk.] and related species. A method for the DNA extraction is described which overcomes these problems.     

Cooperative enhancement at the Drosophila Sgs-3 locus

The Drosophila glue gene Sgs-3 is specifically expressed in the secretory cells of the salivary glands of third instar larvae. We have assayed the expression of gene fusions to determine the role of cis-acting Sgs-3 sequences in conferring this pattern of expression. These experiments define two regulatory regions required for expression of reporter genes from the Sgs-3 promoter. One region, between 106 and 56 bp upstream of the Sgs-3 mRNA 5' end is sufficient for low but correct tissue- and stage-specific expression. A second region, lying between 629 and 130 bp 5' of the RNA start site is functionally equivalent; that is, it alone will also direct low level, specific expression. These two regions act synergistically to give high level expression. More distant upstream regions function to further increase levels of expression. These two regulatory elements can confer a salivary gland-specific pattern of expression on a heterologous promoter and are also sufficient to drive gene expression in other Drosophila species, implying conservation of regulators.     

THE SPORE-GERMINATION PATTERN OF THELYPTEROID FERNS

Cell division patterns in germinating spores of several Thelypteris species were studied using light microscopy of sectioned material and scanning electron microscopy. All species exhibited the same basic germination pattern, characterized by an asymmetric cell division of the spore parallel to the equatorial plane to delimit a proximal rhizoid, followed by a perpendicular division of the basal cell to form the protonemal cell. While spore-germination patterns appear to be a potentially useful taxonomic character in some groups of ferns, the homogeneity in this character exhibited by the thelypteroid group impairs its usefulness in the taxonomy of Thelypteris.     

Length–weight relationships of six indigenous fish species from the River Cauvery and its estuary,India

The present study estimated length–weight relationships (LWRs) for six indigenous fish species (Barilius gatensis, Salmostoma acinaces, S. boopis, Puntius amphibius, Hemibagrus punctatus and Ambassis miops) based on specimens collected from River Cauvery (including estuary) during July 2017–January 2020. The sampling surveys were carried out in three distinct sampling seasons, viz., the pre-monsoon (March–May), the monsoon (July–October) and the post-monsoon (November–February). Majority of the fish specimens dealt in the study were collected from multi-meshed monofilament gill nets (mesh sizes 18, 30, 45, 60, 90, 110, 120 and 150 mm) operated by local fishers. For those sites situated in the protected areas, sampling was carried out by cast nets with prior permission from the local administration and the collected fishes were released back into river after length–weight measurements. The length measurements were noted as total length (TL) measured to the nearest 0.1 cm by using a digital Vernier caliper. A digital balance was used for weight measurements with an accuracy of 0.01 g. The study recorded a new maximum length of 48 cm for H. punctatus. The LWR data generated from the present study are significant for proper assessment of the stock status and their management, if collected together with other essential biological and physical parameters.     

A comparative study on the cellular stressors in mesenchymal stem cells (MSCs) and pancreatic β-cells under hyperglycemic milieu

Molecular and Cellular Biochemistry - β-cell dysfunction is a critical determinant for both type 1 diabetes and type 2 diabetes and β-cells are shown to be highly susceptible to cellular...     

Mechanism of mutant calreticulin-mediated activation of the thrombopoietin receptor in cancers

Myeloproliferative neoplasms (MPNs) are frequently driven by mutations within the C-terminal domain (C-domain) of calreticulin (CRT). CRT_Del52 and CRT_Ins5 are recurrent mutations. Oncogenic transformation requires both mutated CRT and the thrombopoietin receptor (Mpl), but the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. We show that the acquired C-domain of CRT_Del52 mediates both Mpl binding and disulfide-linked CRT_Del52 dimerization. Cysteine mutations within the novel C-domain (C400A and C404A) and the conserved N-terminal domain (N-domain; C163A) of CRT_Del52 are required to reduce disulfide-mediated dimers and multimers of CRT_Del52. Based on these data and published structures of CRT oligomers, we identify an N-domain dimerization interface relevant to both WT CRT and CRT_Del52. Elimination of disulfide bonds and ionic interactions at both N-domain and C-domain dimerization interfaces is required to abrogate the ability of CRT_Del52 to mediate cell proliferation via Mpl. Thus, MPNs exploit a natural dimerization interface of CRT combined with C-domain gain of function to achieve cell transformation.     

Synconset Waves and Chains: Spiking Onsets in Synchronous Populations Predict and Are Predicted by Network Structure

Synfire waves are propagating spike packets in synfire chains, which are feedforward chains embedded in random networks. Although synfire waves have proved to be effective quantification for network activity with clear relations to network structure, their utilities are largely limited to feedforward networks with low background activity. To overcome these shortcomings, we describe a novel generalisation of synfire waves, and define ‘synconset wave’ as a cascade of first spikes within a synchronisation event. Synconset waves would occur in ‘synconset chains’, which are feedforward chains embedded in possibly heavily recurrent networks with heavy background activity. We probed the utility of synconset waves using simulation of single compartment neuron network models with biophysically realistic conductances, and demonstrated that the spread of synconset waves directly follows from the network connectivity matrix and is modulated by top-down inputs and the resultant oscillations. Such synconset profiles lend intuitive insights into network organisation in terms of connection probabilities between various network regions rather than an adjacency matrix. To test this intuition, we develop a Bayesian likelihood function that quantifies the probability that an observed synfire wave was caused by a given network. Further, we demonstrate it''s utility in the inverse problem of identifying the network that caused a given synfire wave. This method was effective even in highly subsampled networks where only a small subset of neurons were accessible, thus showing it''s utility in experimental estimation of connectomes in real neuronal-networks. Together, we propose synconset chains/waves as an effective framework for understanding the impact of network structure on function, and as a step towards developing physiology-driven network identification methods. Finally, as synconset chains extend the utilities of synfire chains to arbitrary networks, we suggest utilities of our framework to several aspects of network physiology including cell assemblies, population codes, and oscillatory synchrony.     

Calreticulin enhances the secretory trafficking of a misfolded α-1-antitrypsin

α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.