Thyroxine-binding globulin: investigation of microheterogeneity

Preparations of T4-binding globulin (TBG) from human serum was performed using only two affinity chromatography steps. Purity of the protein was demonstrated by a single band in overloaded disc and sodium dodecyl sulfate electrophoresis, equimolar binding to T4, and linearity in sedimentation velocity run. The molecular weight was calculated to be 60,000 +/- 3,000 daltons (n = 3), the sedimentation coefficient was 3.95S, and the Stokes' radius was 37 A. The amino acid composition was found to be in good agreement with the calculations of other authors. By isoelectric focussing (IEF), pure TBG showed four main bands at pH 4.25, 4.35, 4.45, and 4.55 together with several fainter bands. The N- acetylneuraminic acid (NANA) content of the four TBG bands isolated by preparative IEF was found to decrease from 10.2 mol NANA/mol TBG in the band at pH 4.25 to 4.8 mol NANA/mol TBG in the band at pH 4.55. No significant difference in the affinity constants of the TBG bands to T4 was found. The affinity constants for TBG ranged from 3.1 x 10(9) to 7.2 x 10(9) M-1. Sequential kinetic desialylation of pure TBG resulted in a progressive tendency toward one major band at pH 6.0. In native sera, microheterogeneity of TBG was detected after IEF on polyacrylamide gel plates by immunofixation. The typical TBG patterns shown by pure TBG were also found in normal subjects. Characteristic deviations from this pattern were found in the sera of females during estrogen therapy or pregnancy, where there was a gradual increase in density of the band at pH 4.25 and the appearance of an additional band at pH 4.15. In sera from patients with liver disease and elevated TBG levels, there was a fading of the acidic bands, whereas the more alkaline band at pH 4.55 was intensified. It is therefore proposed that microheterogeneity of TBG is caused by differences in NANA content and that variations of TBG patterns in native sera may reflect altered TBG synthesis or degradation. A genetically related microheterogeneity of TBG could not be demonstrated after examination of 800 sera, including 2 families with quantitative TBG deficiency

Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

Ubiquitin-dependent sorting of membrane proteins in endosomes directs them to lysosomal degradation. In the case of receptors such as the epidermal growth factor receptor (EGFR), lysosomal degradation is important for the regulation of downstream signalling. Ubiquitinated proteins are recognised in endosomes by the endosomal sorting complexes required for transport (ESCRT) complexes, which sequentially interact with the ubiquitinated cargo. Although the role of each ESCRT complex in sorting is well established, it is not clear how the cargo is passed on from one ESCRT to the next. We here show that flotillin-1 is required for EGFR degradation, and that it interacts with the subunits of ESCRT-0 and -I complexes (hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Tsg101). Flotillin-1 is required for cargo recognition and sorting by ESCRT-0/Hrs and for its interaction with Tsg101. In addition, flotillin-1 is also required for the sorting of human immunodeficiency virus 1 Gag polyprotein, which mimics ESCRT-0 complex during viral assembly. We propose that flotillin-1 functions in cargo transfer between ESCRT-0 and -I complexes

Application of the analytic hierarchy process in a comparative analysis of automated information systems

In a scientific study, we investigated decision-making methods, in particular, the methods of expert estimations to solve problems. Reflected the possibility of using the analytic hierarchy process for researching and selection of information systems in accordance with the requirements of the customer. Comparative analysis has been performed on the example of automated library information systems

Platelet migration and bacterial trapping assay under flow

Blood platelets are critical for hemostasis and thrombosis, but also play diverse roles during immune responses. We have recently reported that platelets migrate at sites of infection in vitro and in vivo. Importantly, platelets use their ability to migrate to collect and bundle fibrin (ogen)-bound bacteria accomplishing efficient intravascular bacterial trapping. Here, we describe a method that allows analyzing platelet migration in vitro, focusing on their ability to collect bacteria and trap bacteria under flow

New insight into breast cancer cells involving drug combinations for dopamine and serotonin receptors

The breast cancer therapies available are insufficient, especially since first-line treatments, such as paclitaxel, result in drug resistance and their toxicity often limits their concentration. Strategies like drug repurposing are beneficial, and novel treatments can emerge by repurposing drugs that interfere with the dopamine and serotonin receptors, and thus influence tumor growth. In this study, the MTT assay was used to test the efficacy of such repurposed drugs commonly used for neurodegenerative disorders that act on the dopamine and serotonin receptors to reduce the MCF7 cell’s viability, either by their single use or in combination with the reference drug paclitaxel. Furthermore, the expression of vimentin and E-cadherin was assayed by immunofluorescence. The dopamine receptor-altering drugs benztropine and thioridazine resulted in the strongest reduction of cell viability when combined with paclitaxel, which may be connected to the alteration of E-cadherin rather than vimentin expression. More studies are needed to understand the mechanism of action of the combinations tested and the efficacious role of dopamine and serotonin.This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) and FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the COMPETE 2020 Operational Programme for Competitiveness and Internationalisation (POCI), Portugal, in the framework of the project IF/00092/2014/CP1255/CT0004. N.V. thanks Fundação para a Ciência e a Tecnologia (FCT, Portugal) for supporting these studies through nationally-funded projects within the CINTESIS R&D unit (reference UIDB/4255/2020). The contents of this report are solely the responsibility of the authors and do not necessarily represent the official view of the FCT

The long noncoding RNA neuroLNC regulates presynaptic activity by interacting with the neurodegeneration-associated protein TDP-43

The cellular and the molecular mechanisms by which long noncoding RNAs (lncRNAs) may regulate presynaptic function and neuronal activity are largely unexplored. Here, we established an integrated screening strategy to discover lncRNAs implicated in neurotransmitter and synaptic vesicle release. With this approach, we identified neuroLNC, a neuron-specific nuclear lncRNA conserved from rodents to humans. NeuroLNC is tuned by synaptic activity and influences several other essential aspects of neuronal development including calcium influx, neuritogenesis, and neuronal migration in vivo. We defined the molecular interactors of neuroLNC in detail using chromatin isolation by RNA purification, RNA interactome analysis, and protein mass spectrometry. We found that the effects of neuroLNC on synaptic vesicle release require interaction with the RNA-binding protein TDP-43 (TAR DNA binding protein-43) and the selective stabilization of mRNAs encoding for presynaptic proteins. These results provide the first proof of an lncRNA that orchestrates neuronal excitability by influencing presynaptic function