SERPINA1 long transcripts produce non-secretory alpha1-antitrypsin isoform: In vitro translation in living cells.

SERPINA1 is a well-studied serpin gene due to its dramatic impact on human health. Translation initiation at the main SERPINA1 start codon produces the only known alpha1-antitrypsin (AAT) isoform intended for secretion. AAT performs essential functions by inhibiting proteases and modulating immunity. However, SERPINA1 expression at the level of translation is not sufficiently studied. Here we hypothesize that the main SERPINA1 ORF can be alternatively translated, producing a non-secretory AAT isoform by either masking or excluding a signal peptide. We defined SERPINA1 long mRNA isoforms specific for prostate (DU145) and liver (HepG2) cell lines and studied their individual expression by in vitro assay. We found that all long transcripts produce both glycosylated secretory AAT-eGFP fusion protein and non-glycosylated intracellular AAT-eGFP (initiated from an alternative AUG-2 start codon), with the proportion regulated by the SERPINA1 5'-UTR. Both fusion proteins localize to distinct cellular compartments: in contrast to a fusion with the secretory AAT accumulating in the ER, the intracellular one exhibits nuclear-cytoplasmic shuttling. We detected putative endogenous AAT isoform enriching the nuclear speckles. CONCLUSION: Alternative translation initiation might be a mechanism through which SERPINA1 expands the biological diversity of its protein products. Our findings open up new prospects for the study of SERPINA1 gene expression.

Towards unveiling the nature of short SERPINA1 transcripts: Avoiding the main ORF control to translate alpha1-antitrypsin C-terminal peptides.

Alternative ORFs in-frame with the known genes are challenging to reveal. Yet they may contribute significantly to proteome diversity. Here we focused on the individual expression of the SERPINA1 gene exon 5 leading to direct translation of alpha1-antitrypsin (AAT) C-terminal peptides. The discovery of alternative ways for their production may expand the current understanding of the serpin gene's functioning. We detected short transcripts expressed primarily in hepatocytes. We identified four variants of hepatocyte-specific SERPINA1 short transcripts and individually probed their potential to be translated in living cells. The long mRNA gave the full-length AAT-eGFP fusion, while in case of short transcripts we deduced four active SERPINA1 in-frame alternative ORFs encoding 10, 21, 153 and 169 amino acids AAT C-terminal oligo- and polypeptides. Unlike secretory AAT-eGFP fusion exhibiting classical AAT behavior, truncated AAT-fusions differ by intracellular retention and nuclear enrichment. Immunofluorescence on the endogenous AAT C-terminal epitope showed its accumulation in the cell nucleoli, indicating that short transcripts may be translated in vivo. FANTOM5 CAGE data on SERPINA1 suggest that short transcripts originate from the post-transcriptional cleavage of the spliced mRNA, initiated mainly from the hepatocyte-specific promoter. CONCLUSION: Short SERPINA1 transcripts may represent a source for the direct synthesis of AAT C-terminal peptides with properties uncommon to AAT.

[Methods of Evaluating the Efficiency of CRISPR/Cas Genome Editing].

The CRISPR/Cas system is currently widely used for genome editing. The procedure of genome editing includes two necessary steps: (i) searching for the most effective guide RNA, and (ii) analyzing clones for presence of the desired mutation. This review presents the methods used to assess the efficiency of the CRISPR/Cas system and to confirm mutation in the target locus and discusses their advantages and disadvantages. It aims to provide information that could help researchers to choose a technique most appropriate for their specific tasks and available resources.

MUC1 in Cancer Immunotherapy - New Hope or Phantom Menace?

Understanding of the functioning of MUC1 (human mucin) has advanced significantly over 40 years of its investigation. The anti-adhesive properties of the extracellular domain, which were the main focus of early studies initially explaining overexpression of MUC1 in progressing oncological diseases, were gradually put on the back burner. Researchers became more interested in its regulatory and signaling functions in cells rather in its anti-adhesive properties. The found the ability of MUC1 for signal transduction, and its ability to participate in cell metabolism opened new possibilities for improved control over cancer cells in addition to just attracting antigens of the immune system to a target. Nevertheless, there are issues in the functioning of MUC1 that raise doubts about its effectiveness in cancer immunotherapy.

Analysis of In Vivo Activity of the Bovine Cholesterol Hydroxylase/Lyase System Proteins Expressed in Escherichia coli.

The cholesterol hydroxylase/lyase (CHL) system, located in the mitochondria of the mammalian adrenal cortex cells, consists of cytochrome P450scc (CYP11A1), adrenodoxin (Adx), and adrenodoxin reductase (AdR) and performs the first stage of the steroidogenesis: AdR and Adx enable the electron transfer between NADPH and cytochrome P450scc, and P450scc catalyzes the conversion of cholesterol into pregnenolone. CHL system was reconstructed in Escherichia coli using the polycistronic plasmid pTrc99A/CHL. In E. coli cells, the recombinant proteins form the catalytically active system. CHL activity towards 22R-hydroxycholesterol was 4.0 ± 1.3 nmol pregnenolone/h per 1 mg homogenate protein. The alteration of the order of heterologous cDNAs in the expression cassette from AdR-Adx-P450scc to P450scc-Adx-AdR results in alteration of stoichiometric ratio P450scc/Adx/AdR from 1:1.45:4.2 to 1:1.67:0.98; the former ratio is more optimal for the functioning of the cytochrome P450scc. The application of modified cDNA of Adx (AdxS112W) does not increase the CHL activity; however, the introduction of the second copy of AdxS112W gene into the expression cassette increases both the expression level of АdxS112W and the CHL activity in comparison with P450scc/АdxS112W/AdR system. In vivo activity of the CHL system in bacteria is limited by the substrate uptake by bacterial cells: it varied in the range of 0.05-0.62 mg pregnenolone/l resting cell suspension per 1-day cultivation, depending on the type and concentration of permeabilizing agents in the medium. The obtained results contribute to the knowledge of CHL system functioning in living bacteria.

Dual Role of the Extracellular Domain of Human Mucin MUC1 in Metastasis.

Human mucin MUC1 plays an important role in cancer development. The increased level of this molecule expression during cancer cell progression induces metastasis and is associated with poor prognosis for patients. There is a large body of experimental data on the role of various functional domains of human mucin MUC1 in metastasis. While, the cytoplasmic domain determined to play a definitive role, the influence of extracellular domain on cancer cell invasiveness still remains unclear. The present paper reveals that the extracellular domain of MUC1 molecule consists of two functional subdomains-the region of tandem repeats (TR) and the region of irregular repeats (IR). We demonstrate the ability of each of these subdomains to alter the invasiveness of cancer cells. The presence of the MUC1 molecules containing TR subdomain (MUC1-TR) on the surface of low-invasive cancer cells leads to the increase in their transendothelial migration potency, while the addition of the IR subdomain to the MUC1-TR molecule (MUC1-IR-TR) restores their natural low invasiveness. J. Cell. Biochem. 118: 4002-4011, 2017. © 2017 Wiley Periodicals, Inc.

Cell Models for the Investigation of the Role of the Mucin MUC1 Extracellular Domain in Metastasizing.

The speculations on the role of MUC1, a substance which is overexpressed in glandular cancer cells, on the metastatic potential of such cells are rooted in data that seem to indicate that cell malignization correlates with a change from the apical localization of mucin MUC1 to a peripheral one. Nonetheless, the role of MUC1 in cancer metastasizing remains far from clear. The major hurdle remains the absence of adequate cell models. The aim of the present study was to create cell models that present different fragments of the human mucin MUC1 extracellular domain on their surface. Genetic constructions were generated on the basis of the plasmid vector pEGFP-N3. These constructions contain fusion genes coding for chimeric proteins composed of different combinations of mucin MUC1 functional domains and identification markers (FLAG-epitope, located at the N-terminus, and EGFP, located at the C-terminus of the chimeric proteins). These constructions were used for a stable transformation of HT-29 human cancer cells. The transformants obtained were characterized by flow cytometry. The low expression level of endogenous mucin MUC1 and the high expression level of recombinant proteins were confirmed by real-time PCR. The microscopic examination of the transformed cells confirmed the membrane localization of the fusion proteins. The resulting cell models could be used to investigate the role of the mucin MUC1 domains in cancer cell metastasizing. The obtained cells are used as an applicable model of MUC1-expressing cancers and might be used to study the role of different functional fragments of mucin MUC1 in metastasizing.

[Fulminant acute pancreatitis. Are there objective criteria of the disease?].

A retrospective analysis has been made of the results of the diagnosis and treatment of 71 patients having severe pancreatitis. As a result, factors were revealed which restrict the possibilities of conservative treatment associated with pessimistic prognosis of the disease. Among them there are pancretitis-specific organic abnormalities (pulmonary, cardio-vascular, renal dysfunctions), diffused pancreonecrosis and infection of destructive zones. All the parameters in question are included in the proposed model of prognosis of the fulminant course of pancreatitis having high diagnostic accuracy up to 88.3%.

Surgical Approach to Treatment of Necrotizing Pancreatitis: Early Primary Drainage without Necrosectomy. Review of Seven Recent Cases.

Unsatisfactory results of surgery in the late course of pancreatic necrosis made us search for indications and variants of operation in the early phase of the disease. As early surgical intervention, the universal approach was used in 7 patients with necrotizing pancreatitis who had a different prevalence of the inflammatory process in the retroperitoneal space. The drainage proved to be effective and enabled us to always prevent generalized infectious complications in the later phases of the disease in absence of local complications specific for open surgery: bleeding and digestive fistulas. In spite of obvious infected process development in primary open surgery, we noticed a stable decrease in procalcitonin level following the drainage. A surgical intervention has been developed enabling one to reveal in time the volume of damaged retroperitoneal fat tissue and to drain it adequately in compliance with the process prevalence, thus avoiding septic complications in the late phase of the disease. The method's advantage involves refusal from necrosectomy in primary intervention, weekly staged revisions of the retroperitoneal space via formed contrapertures as dictated by evolution of the necrotic process in the gland.

[Effects of Helicobacter pylori on the results of conservative surgery in duodenal ulcer]. / Vliianie Helicobacter pylori na rezul'taty organosokhraniaiushchikh operatsii pri iazvennoi bolezni dvenadtsatiperstnoi kishki.

Sixty-two patients with exacerbation of duodenal ulcer were tested for Helicobacter pylori (HP). Bacterial invasion was found in 54 out 62 patients (87%). The degree of bacterial dissemination, the physiological resistance of the tegmental epithelium, and granulocytic infiltration of the mucosa were studied one and three months after selective proximal vagotomy (SPV) in 16 patients with HP. The control group consisted of seven patients without HP. It was found that bacterial invasion led to diminution of the physiological resistance of the tegmental epithelium and caused a chronic inflammatory process in the mucosal stroma. Bacterial dissemination after the operation not only corresponded to its initial level in the duodenum but tended to increase in the antrum and fundus of the stomach.