[Landscape of KRAS, BRAF, and PIK3CA Mutations and Clinical Features of EBV-Associated and Microsatellite Unstable Gastric Cancer].

Personalization of gastric cancer (GC) treatment is an urgent problem because of the clinical heterogeneity and aggressive course of the disease. Four GC subtypes were isolated based on molecular characteristics by The Cancer Genome Atlas researchers in 2014: Epstein-Barr virus positive (EBV^(+)), microsatellite unstable (MSI), chromosomally unstable (CIN), and genomically stable (GS). There is no unified method to detect the CIN and GS subtypes today, while MSI and EBV status assessments are used routinely and are of great clinical importance. A total of 159 GC samples were tested for MSI, EBV DNA, and somatic mutations in codons 12-13 (exon 2), 61 (exon 3), and 146 (exon 4) of the KRAS gene; codons 597-601 (exon 15) of the BRAF gene; and codons 542-546 (exon 9), 1047-1049 (exon 20) of the PIK3CA gene. EBV^(+) GC was detected in 8.2% of samples; and MSI, in 13.2%. MSI and EBV+ were found to be mutually exclusive. The mean ages at GC manifestation were 54.8 and 62.1 years in patients with EBV^(+) and MSI GCs, respectively. EBV^(+) GC affected men in 92.3% of cases, 76.2% of the patients were older than 50 years of age. Diffuse and intestinal adenocarcinomas were diagnosed in 6 (46.2%) and 5 (38.5%) EBV^(+) cases, respectively. MSI GC equally affected men (n = 10, 47.6%) and women (n = 11, 52.4%). The intestinal histological type was the most prevalent (71.4%); the lesser curvature was affected in 28.6% of the cases. The E545K variant of PIK3CA was observed in one EBV^(+) GC case. A combination of clinically significant variants of KRAS and PIK3CA was found in all MSI cases. The BRAF V600E mutation, which is specific to MSI colorectal cancer, was not detected. The EBV^(+) subtype was associated with better prognosis. The five-year survival rates were 100.0 and 54.7% for MSI and EBV^(+) GCs, respectively.

[Surgical treatment of patients with leiomyosarcoma of inferior vena cava]. / Khirurgicheskoe lechenie bol'nykh s leiomiosarkomoi nizhnei poloi veny.

AIM: To evaluate the results of surgical treatment of patients with leiomyosarcoma of inferior vena cava. MATERIAL AND METHODS: For the period 2003-2016 21 patients with leiomyosarcoma of IVC were operated at the abdominal department of Blokhin Russian Cancer Research Center. 23.8% patients (n=5) underwent longitudinal resection of IVC, 23.8% (n=5) - circular resection and ligation of IVC, 23.8% (n=5) - circular resection and ligation of IVC, left renal vein ligation, 9.5% (n=2) - circular resection and replacement of IVC, 9.5% (n=2) - circular resection and replacement of IVC, ligation of left renal vein, 9.5% (n=2) - circular resection and replacement of IVC, left renal vein implantation into the prosthesis. RESULTS: The incidence of postoperative complications was 38% (n=8). Postoperative mortality was 4.8% (n=1). R1-resection was made in 4.8% patients (n=1). 1- and 2-year recurrence-free survival was 67% and 50% respectively. Overall 1- and 2-year survival was 86% and 68% respectively. CONCLUSION: In patients with leiomyosarcoma of inferior vena cava surgery is feasible and safe with satisfactory long-term results.

[Iliac arteries and aortic repair in patients with retroperitoneal sarcoma]. / Plastika podvzdoshnykh arterii i aorty u bol'nykh neorgannoi zabriushinnoi sarkomoi (s kommentariem).

AIM: To evaluate the results of surgical treatment in patients with retroperitoneal sarcoma and invasion into great vessels. MATERIAL AND METHODS: For the period 2009-2016 fourteenth patients with retroperitoneal sarcoma underwent resection and repair of great vessels at the abdominal department of N.N. Blokhin Russian Cancer Research Center. In 12 cases circular resection of infrarenal aorta, aortic bifurcation and iliac arteries followed by their replacement was made. 2 patients underwent circular resection of iliac arteries. RESULTS: Postoperative morbidity was 42.8%. There was no postoperative mortality. 10 of 14 patients are alive from 2 to 70 months after surgery. One patient died in 2 months postoperatively from unknown reasons, 3 patients died in 18, 20 and 30 months respectively due to progression of the disease. CONCLUSION: Overall survival and acceptable surgical risk underline the value of en block resection of retroperitoneal sarcoma together with involvement blood vessels.

Dependence of prePhoA-phospholipid interaction in vivo and in vitro on charge of signal peptide N-terminus and content of anionic phospholipids in membranes.

Replacement of the positively charged signal peptide with neutral or negatively charged peptides due to substitution of Lys(-20) in the N-terminal region of the signal peptide leads to decreases in the rate of prePhoA membrane translocation in vivo and in the efficiency of prePhoA insertion into liposomes in vitro. The effect of anionic phospholipids on prePhoA insertion into model membranes is determined by the signal peptide N-terminus charge, while the dependence of prePhoA translocation across the cytoplasmic membrane in vivo is not, under the studied variations in the content of anionic phospholipids. This is evidence of the possibility of direct electrostatic interaction between the signal peptide N-terminus and anionic phospholipids, which in vivo, however, seems to involve some proteins of the Sec machinery.

The net charge of the first 18 residues of the mature sequence affects protein translocation across the cytoplasmic membrane of gram-negative bacteria.

This statistical study shows that in proteins of gram-negative bacteria exported by the Sec-dependent pathway, the first 14 to 18 residues of the mature sequences have the highest deviation between the observed and expected net charge distributions. Moreover, almost all sequences have either neutral or negative net charge in this region. This rule is restricted to gram-negative bacteria, since neither eukaryotic nor gram-positive bacterial exported proteins have this charge bias. Subsequent experiments performed with a series of Escherichia coli alkaline phosphatase mutants confirmed that this charge bias is associated with protein translocation across the cytoplasmic membrane. Two consecutive basic residues inhibit translocation effectively when placed within the first 14 residues of the mature protein but not when placed in positions 19 and 20. The sensitivity to arginine partially reappeared again 30 residues away from the signal sequence. These data provide new insight into the mechanism of protein export in gram-negative bacteria and lead to practical recommendations for successful secretion of hybrid proteins.

Interaction of mutant alkaline phosphatase precursors with membrane phospholipids in vivo and in vitro.

Positively charged amino acid residues in the N-terminal domain of the signal peptides of secreted proteins are thought to interact with negatively charged anionic phospholipids during the initiation of secretion. To test this hypothesis, substitutions of the uncharged Ala or the negatively charged Glu residue for the positively charged Lys-20 of the N-terminus of the signal peptide of Escherichia coli alkaline phosphatase were introduced using a modified method of oligonucleotide-directed mutagenesis. We found that Lys-20 is involved in the interaction of the signal peptide with anionic phospholipids in vivo and effects the efficiency of insertion of the signal peptide of isolated precursor into model phospholipid membranes in vitro. We also show that the efficiency of signal peptide insertion into the lipid bilayer depends on the fluidity of the bilayer.

Interaction of the E. coli alkaline phosphatase precursor with model phospholipid membranes.

An interaction of a precursor of the E. coli secreted alkaline phosphatase (prePhoA) containing a signal peptide and model bilayer membranes prepared from the lipids of E. coli has been studied. The interaction was evaluated by monitoring of the state of the lipid phase by fluorescence spectroscopy. The role of the signal peptide in this process was evaluated by a comparative study of the interaction of the mature alkaline phosphatase which does not contain the signal peptide with the model membranes. The precursor was shown to be inserted into the lipid bilayer since the fluorescence anisotropy of a hydrophobic probe, diphenylhexatriene, was enhanced. The intensity of the process is determined by the presence of the signal peptide and depends on the pH of the medium. The data indicate that the mature polypeptide chain of the enzyme also has affinity for the membrane.

Positively charged lysine at the N-terminus of the signal peptide of the Escherichia coli alkaline phosphatase provides the secretion efficiency and is involved in the interaction with anionic phospholipids.

Positively charged amino acid residues at the N-terminus of the signal peptide (SP) have been proposed to play a significant role in the initial step of protein secretion in bacteria. To test this hypothesis, Lys(-20) of the Escherichia coli alkaline phosphatase SP was replaced by other amino acid residues, and the effect of these substitutions on protein maturation was studied. The introduction of negatively charged and hydrophobic amino acids resulted in a decrease in secretion efficiency and impaired the SP-APL interaction, whereas His and Tyr had no significant effect. A structural analysis of the SP-APL interaction suggests that the positively charged Lys(-20) determines the stability of the complex.

[Disruption of processing of alkaline phosphatase as a result of single amino acid changes affects the composition and metabolism of phospholipids from Escherichia coli, secreting mutant proteins]. / Narushenie protsessinga shchelochnoi fosfatazy kak sledstvie edinichnykh zamen aminokislot, vliiaet na sostav i obmen fosfolipidov kletok Escherichia coli, sekretiruiushchikh mutantnye belki.

Amino acid substitutions in the cleavage site of the E. coli alkaline phosphatase signal peptide Val for Ala(-1) or Pro for Arg(+1) result in the block of the enzyme processing. In cells secreting such mutant proteins the relative content and rate of turnover of anionic phospholipids (phosphatidylglycerol and cardiolipin) are increased. The rise of the transfer of the phosphoglycerol residue from phosphatidylglycerol to periplasmic membrane derived oligosaccharides or to the model substrate, arbutin performed by the activity of phosphoglycerol transferase I testifies to phosphatidylglycerol accumulation on the outer surface of the cytoplasmic membrane. The results suggest of phosphatidylglycerol interaction with the alkaline phosphatase precursor and their subsequent joint translocation through the cytoplasmic membrane of E. coli.

[Isolation and certain properties of mutant alkaline phosphatase of Escherichia coli]. / Vydelenie i nekotorye svoistva mutantnykh shchelochnykh fosfataz Escherichia coli.

Natural and mutant alkaline phosphatases with amino acid substitutions in the processing site and N-terminal domain of the mature polypeptide chain Val for Ala(-1), Gln for Glu (+4) and simultaneously Gln for Glu (+4) and Ala for Arg (+1) have been isolated from the periplasm and cultural fluid of E. coli. It has been found that these substitutions have little effect on the dependence of the enzyme activity on pH, ionic strength and temperature but influence its isoenzymic spectrum and decrease (almost twofold) the maximal rate of the enzyme-catalyzed reaction. Extracellular enzymes display, in contrast with periplasmic ones, other catalytic properties (Vmax) and binding activity (Km). After translocation through the outer membrane all the enzymes display decreased Vmax and increased Km. These changes are especially well-pronounced in case of the mutant protein PhoA46 which contains an uncleaved signal peptide due to the impossibility of processing resulting from the substitution of Val for Ala(-1). The Vmax for this protein is decreased 20 times, while the Km is increased 4-fold. The protein also shows a higher (in comparison with other proteins) sensitivity towards proteolytic enzymes and is less resistant upon storage. The experimental data suggest that the changes in the N-end of alkaline phosphatase located at a long distance from its active center influence the enzyme function.

[Biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. II. Effect of replacing amino acids at the processing site and N-terminal domain of the mature polypeptide chain of alkaline phosphatase on its biogenesis]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykh form u Escherichia coli. II. Vliianie zamen aminokislot v saite protsessinga i N-kontsevom domene zreloi polipeptidnoi tsepi shchelochnoi fosfatazy na ee biogenez.

The effect of amino acid substitutions in E. coli alkaline phosphatase on its biogenesis has been studied. The substitution of Val for Ala(-1) in the signal peptide cleavage site completely inhibits all stages of posttranslational modification: processing and formation of isozymes. The absence of processing does not prevent translocation of the precursor across the cytoplasmic membrane and formation of an active enzyme macromolecule. The precursor of the above mutant protein was found in the periplasm and in the cytoplasmic membrane. The substitution of Gln for Glu(+4), as well as the double substitution of Ala for Arg(+1) and Gln for Glu(+4), in the N-terminus of mature polypeptide chain result in the change in the isozyme spectrum. Differences in the rates of processing in vivo of both mutant proteins were not revealed. However, the double amino acid substitution significantly increases the efficiency of in vitro processing. All amino acid substitutions studied have no effect on the peculiarities of biogenesis which are conditioned by oversynthesis of the enzyme encoded by the phoA gene in the plasmid: secretion into the culture medium and accumulation of precursor as insoluble aggregates in the cytoplasm. However, extracellular activities of mutant proteins differ from that of the wild-type protein, which may result from the change either in the efficiency of their secretion or in their catalytic properties.

[Biogenesis and secretion of alkaline phosphatase and its mutants in Escherichia coli. III. Substitution of N-terminal amino acids of alkaline phosphatase affect its biogenesis]. / Izuchenie biogeneza i sekretsii shchelochnoi fosfatazy i ee mutantnykhform u Escherichia coli. III. Zameny N-kontsevoi aminokisloty shchelochnoi fosfatazy vliiaiut na ee biogenez.

The effect of the N-terminal amino acid substitution on E. coli alkaline phosphatase biogenesis has been studied. The substitutions of Ser, Gln, Tyr, Leu, Gly, Ala, Glu, Phe, His, Cys, Lys and Pro for Arg(+1) were obtained by creating amber mutation at the corresponding position within phoA gene and expressing this mutated gene in E. coli strains that produce the amber-suppressor tRNAs. All mutant proteins were shown to translocate across the cytoplasmic membrane and possess enzyme activity. The introduction of Pro in +1 position disturbs the cleavage of signal peptide whereas the insertion of the other amino acids does not change the rates of processing in comparison with wild-type protein. All amino acid substitutions affect alkaline phosphatase isoenzyme composition. Some experimental evidence were also obtained on the specificity of protease, which split off N-terminal Arg during alkaline phosphatase maturation.