Application of RNA subcellular fraction estimation method to explore RNA localization regulation

RNA localization is involved in multiple biological processes. Recent advances in subcellular fractionation-based sequencing approaches uncovered localization pattern on a global scale. Most of existing methods adopt relative localization ratios (such as ratios of separately normalized transcripts per millions of different subcellular fractions without considering the difference in total RNA abundances in different fractions), however, absolute ratios may yield different results on the preference to different cellular compartment. Experimentally, adding external Spike-in RNAs to different fractionation can be used to obtain absolute ratios. In addition, a spike-in independent computational approach based on multiple linear regression model can also be used. However, currently, no custom tool is available. To solve this problem, we developed a method called subcellular fraction abundance estimator to correctly estimate relative RNA abundances of different subcellular fractionations. The ratios estimated by our method were consistent with existing reports. By applying the estimated ratios for different fractions, we explored the RNA localization pattern in cell lines and also predicted RBP motifs that were associated with different localization patterns. In addition, we showed that different isoforms of same genes could exhibit distinct localization patterns. To conclude, we believed our tool will facilitate future subcellular fractionation-related sequencing study to explore the function of RNA localization in various biological problems.

Turning the spotlight on the oligosaccharide chain of GM1 ganglioside

It is well over a century that glycosphingolipids are matter of interest in different fields of research. The hydrophilic oligosaccharide and the lipid moiety, the ceramide, both or separately have been considered in different moments as the crucial portion of the molecule, responsible for the role played by the glycosphingolipids associated to the plasma-membranes or to any other subcellular fraction. Glycosphingolipids are a family of compounds characterized by thousands of structures differing in both the oligosaccharide and the ceramide moieties, but among them, the nervous system monosialylated glycosphingolipid GM1, belonging to the group of gangliosides, has gained particular attention by a multitude of Scientists. In recent years, a series of studies have been conducted on the functional roles played by the hydrophilic part of GM1, its oligosaccharide, that we have named “OligoGM1”. These studies allowed to shed new light on the mechanisms underlying the properties of GM1 defining the role of the OligoGM1 in determining precise interactions with membrane proteins instrumental for the neuronal functions, leaving to the ceramide the role of correctly positioning the GM1 in the membrane crucial for the oligosaccharide-protein interactions. In this review we aim to report the recent studies on the cascade of events modulated by OligoGM1, as the bioactive portion of GM1, to support neuronal differentiation and trophism together with preclinical studies on its potential to modify the progression of Parkinson’s disease.

PECULIARITIES OF THE EFFECTS OF BILE ACIDS ON ATPASE ACTIVITY OF THE COLON MUCOSA IN PATIENTS WITH OVERWEIGHT AND IRRITABLE BOWEL SYNDROME

The aim is to investigate the effect of bile acids on the ATPase activity of the colon mucosa in patients with overweight and irritable bowel syndrome (IBS). Materials and methods: Completely examined 12 patients with IBS and overweight.We estimated the ATPase activity of colon mucous of the patients with IBSspectrophotometrically by determined the content of orthophosphate that was released after ATP hydrolysis. We studied the effect of 3-sulphate of taurolitocholate (TLC-S) on specific activities of Na+/ K+-ATPase, Ca2+-ATPase of endoplasmatic reticulum (EPR),Ca2+-ATPase of plasmatic membrane (PM) and basal Mg2+-ATPase of postmitochondrial subcellular fraction of colon mucous of the patients with IBS. Results: We establishedthe specific activities of Na+/K+-ATPase, Ca2+-ATPase of EPR,Ca2+-ATPase of PM and basal Mg2+-ATPase. Therewere(6.06 ± 1.61), (5.88 ± 1.19), (8.86 ± 1.56) (6.44 ± 2.02)μmol Pi/ mg protein per hour, respectively. TLC-S (50 μM) did not causedany change of Na+/K+-ATPase , as well as Ca2+-ATPasesactivities, but statistically significant increased activity of Mg2+-ATPase of postmitochondrial subcellular fraction of colon mucous of the patients with IBS by 4 fold. Conclusions: TLC-S increased basal Mg2+-ATPase in the postmitochondrial fraction of colon mucous of the patients with overweight and IBS, but had no effect on Na+/K+-ATPase and Ca2+-ATPases activities. It has been suggested that activation of basal Mg2+-ATPase under by TLC-S may indicates the role of the endo-lysosomal system of epitheliocytes of colon mucous in developing of pathology IBS.

The effect of calcium to magnesium ratio on adenosine triphosphatases from wheat roots

The activities of ATPase isolated from 4 days old wheat roots grown on distilled water were found to be associated with the ratio of Ca : Mg ions added in different proportions to the incubation medium. This relationship was stated in the crude cell wall free homogenate (fraction I) as well as subcellular fraction II and fraction III under investigations. It is stated moreover that activity of ATPases extracted from wheat roots grown on deficient nutrient solution with different Ca : Mg ratio is dependend on exogenic ratio of these cations. These results support the concept that activity of plant ATPases depends not only .on mineral nutrition and concentrations of Ca²⁺ and Mg²⁺ ions but also on the Ca : Mg ratio in soil or in nutrient solution and in separate subcellular fractions of plant cells.

Characterization of Ca2+ uptake in a subcellular membrane fraction of Herpetomonas sp. promastigotes

SUMMARYATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0·5 of 0·1 μm and a Hill number (nH)=2·8±0·4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7·0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0·25±0·19 μm and Km2=29·6±6·8 μm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.