Skin autofluorescence and peripheral neuropathy four years later in type 1 diabetes.

OBJECTIVE: Advanced glycation end products (AGEs) are involved in diabetes complications. We aimed to investigate whether the accumulation of AGEs measured by skin autofluorescence (sAF) was associated with signs of diabetic peripheral neuropathy and to sensitivity, pain, motor and autonomic function 4 years later in patients with type 1 diabetes. METHODS: At baseline, 188 patients (age 51 years, diabetes duration 22 years) underwent skin autofluorescence measurement using the AGE Reader. Four years later, signs of diabetic peripheral neuropathy were defined as the presence of neuropathic pain and/or feet sensory loss or foot ulceration. Neurological tests were systematically performed: vibration perception threshold by neuroesthesiometry, neuropathic pain by the Douleur Neuropathique en 4 Questions score, muscle strength by dynamometry and electrochemical skin conductance. Multivariate analyses were adjusted by age, sex, height, body mass index, tobacco, HbA1c , diabetes duration, estimated glomerular filtration rate and albumin excretion rate. RESULTS: At the 4-year follow-up, 13.8% of patients had signs of diabetic peripheral neuropathy. The baseline sAF was higher in those with signs of diabetic peripheral neuropathy (2.5 ± 0.7 vs 2.1 ± 0.5 arbitrary units (AU), p < 0.0005). In the multivariate analysis, a 1 SD higher skin autofluorescence at baseline was associated with an increased risk of signs of neuropathy (OR = 2.68, p = 0.01). All of the neurological tests were significantly altered in the highest quartile of the baseline sAF (>2.4 AU) compared with the lowest quartiles after multivariate adjustment. CONCLUSION: This non-invasive measurement of skin autofluorescence may have a value for diabetic peripheral neuropathy in type 1 diabetes and a potential clinical utility for detection of diabetic peripheral neuropathy. Copyright © 2016 John Wiley & Sons, Ltd.

[Genetic effects of bystander factors from the blood sera of people irradiated as the result of the Chernobyl accident].

The purpose of this work was the analysis of the effects of bystander factors from blood sera of people affected by the Chernobyl accident on human keratinocyte cell culture (HPV-G cells). A new method was developed for evaluation of the bystander factor presence in vivo in blood of the people irradiated by the Chernobyl accident. Affected population groups included liquidators of the Chernobyl accident and people living and working in areas of the Gomel region contaminated by radionuclides. The analysis has shown that bystander factors persist in Chernobyl liquidator blood samples for more than 20 years since irradiation. The data suggest that blood sera contain bystander factors, which are able to induce micronuclei and decrease the metabolic activity of HPV-G cells.

[Problems of ante mortem diagnostics of prion diseases].

The review presents the state-of-the-art on the problem of diagnosis of prion diseases (PD) in humans and animals with a brief description of their etiology and pathogenesis. We pointed out that understanding the nature of the etio logical agent of PD determined their zoonotic potential and led to the development of highly specific immunological diagnostic methods aimed at identifying the infectious isoform of prion protein (PrPd) as the only marker of the disease. In this regard, we briefly summarize the results of studies, including our own, concerning the conversion of normal prion protein molecules (PrPc) to PrPd, the production of monoclonal antibodies and their application as immunodiagnostic reagents for the post-mortem detection of PrPd in various formats of immunoassay. We also emphasize the issues related to the development of methods for ante mortem diagnostics of PD. In this regard, a method for amplifying amino acid sequences using quacking-induced conversion of PrPc to PrPd in real time (RTQuIC) described in details. The results of recent studies on the assessment of the sensitivity, specificity and reproducibility of this method, carried out in various laboratories around the world, are presented. The data obtained indicate that RT-QuIC is currently the most promising laboratory assay for detecting PrPd in biological material at the preclinical stage of the disease. The significant contribution of US scientists to the introduction of this method into clinical practice on the model of diagnosis of chronic wasting disease of wild Cervidae (CWD) is noted. The possible further spread of CWD in the population of moose and deer in the territories bordering with Russia, as well as the established fact of alimentary transmission of CWD to macaques, indicate the threat of the appearance of PD in our country. In conclusion, the importance of developing new hypersensitive and/or selective components of known methods for PrPd identification from the point of view of assessing the risks of creating artificial infectious prion proteins in vivo or in vitro, primarily new pathogenic isoforms ("strains") and synthetic prions, was outlined.

[Methods for diagnosis of prion diseases].

The epidemic of bovine spongiform encephalopathy (BSE) in the United Kingdom, its related occurrence of a new type of Creutzfeld-Jacob disease and proven cases of this type of the disease transmitted by blood transfusion initiated intensive studies to develop a inexpensive, prompt, and sensitive method for the early lifetime diagnosis of prion diseases. This would permit initiation of the timely treatment of the patients and prevention of contamination of foodstuffs. However, despite significant progress made in this direction, this objective has not yet been achieved. The present review highlights the currently available methods for the diagnosis of transmissible spongiform encephalopathies, as well as the latest developments in the ultrasensitive detection of these diseases, which is based on the misfolded prion protein complex.

[Development of a diagnostic test system on the basis of sandwich ELISA for the detection of avian influenza A virus].

A panel of hybridomas producing monoclonal antibodies (MAbs) to nucleocapsid protein (NP) of avian influenza A virus was obtained. On the basis of 2 MAbs, the authors designed an antigen-bound ELISA (sandwich ELISA), in which NP3 MAbs were used as antigen-bound antibodies and NP MAbs conjugated with horse radish peroxidase as antigen detection antibodies. The specificity of the test system to avian influenza virus was determined. The developed test system was ascertained to specifically detect influenza A virus of all study subtypes and to yield no cross reactions with other tested virus pathogens. The sensitivity of the sandwich ELISA was 30 ng/ml of NP in the urine-treated virus preparations. The assay was tested on experimental H5N1-infected mice. The findings positively correlated with the results of postmortem studies and with the virus isolation method in the chick embryos. The developed test system may be used to detect avian influenza A virus as an alternative or supplement to other diagnostic techniques.

Association of advanced glycation end products and chronic kidney disease with macroangiopathy in type 2 diabetes.

AIMS: Accumulation of advanced glycation end-products (AGEs), may explain the major contribution of chronic kidney disease (CKD) to cardiovascular events in patients with type 2 diabetes (T2D) related to their impaired renal function. The aim of this study was to analyze the factors associated with AGE assessed by skin autofluorescence and their association with macroangiopathy in T2D. METHODS: We measured skin autofluorescence in patients hospitalized for T2D. Glomerular filtration rates were estimated (eGFR) by the EPI-CKD formula. Associations between skin autofluorescence, renal function and macroangiopathy were explored by multivariate analyses adjusting for diabetes duration and control. RESULTS: The 418 patients had T2D since 13.3 (SD 9.8) years on average, high mean HbA1C: 8.9%, (SD 1.8), (74 mmol/mol, (SD 15)) and often renal complications (49.4% with CKD). Their mean skin autofluorescence was 2.53 (SD 0.62) A.U. In multivariate linear regression, skin autofluorescence was significantly associated with age (+0.20 for ten more years, p<0.0001), renal insufficiency (-0.07 for less 10 mL/min/1.73 m² eGFR, p<0.0001) and smoking (+0.21, p=0.0004). Autofluorescence (p=0.01), but not CKD, was associated with macroangiopathy independent of diabetes duration and control. CONCLUSIONS: Accumulation of AGEs is independently associated with renal insufficiency and macroangiopathy in patients with T2D.

[Molecular-genetic status of the adolescents living in the condition of the constant influense of low dose radiation]. / Molekuliarno-geneticheskii status detei i podrostkov, prozhivaiushchikh v usloviiakh khronicheskogo nizkodozorovogo vozdeistviia.

In the present investigation the analysis of the cytogenetic status and some blood characteristics of the adolescents from the contaminated area of Vulka-2 in Belarus has been done. The obtained data testifed that patients living in the condition of chronic radiation influence had essentially increased general frequency of all types of chromosome aberrations. First of all the elevated level of radiation markers is marked (0.54 +/- 0.11 and 0.11 +/- 0.05% in control. group, p < 0.05), the growth of double fragments (1.27 +/- 0.17 and 0.40 +/- 0.09% in control group, p < 0.05). The Spearmen correlation alalysis has revealed statistically significant increase of apoptosis level on the background of increased level of micronuclei (R = 0.832, p < 0.01). Thus our data testify that the degree of the biological homeostasis violation is intimately connected with the level of genome destabilization.

[Activation of viral reproduction in a mixed infection with human immunodeficiency virus and herpes viruses]. / Aktivatsiia reproduktsii virusov pri smeshannoi infektsii virusom immunodefitsita cheloveka i gerpes-virusami.

Mutual activation of reproduction of type 1 HIV and herpes simplex types 1 and 2 viruses (HSV) was observed in simultaneous infection of continuous T-cellular lymphoblastoid lines (CEM, 119, Hut-78, MT-4, Jurkat-tat) and U-937 monocytic line. Syncytium formation and cytodestructive pattern of reproduction of viruses of both families in these cell lines necessitated the use of enzyme immunoassay (EIA) to detect the antigens of these viruses in order to assess the level of reproduction. The concentration of HIV antigens in EIA increased in mixed infection by 1.4 to 2.1 times in different cultures in comparison with the culture infected with HIV-1 alone, and concentrations of HSV-1 and HSV-2 increased by 1.3-1.8 times in mixed infection, in comparison with reproduction in lymphoblastoid cultures infected with HSV alone. EIA was alone used to examine the production of IgG and IgM antibodies to Epstein-Barr virus, another representative of Herpesviridae family, in the blood sera of patients with immunodeficiency states in whose sera antibodies to proteins produced by gag HIV gene (p15/17, p24, p55) were detected. Increased concentration of IgG antibodies were revealed in 36% of these patients, whereas in healthy donors the sera with elevated concentrations of IgG to Epstein-Barr virus were far less incident (12%). A hypothesis about mutual activation of HIV and herpes viruses is put forward.

[Use of recombinant protein E2 of the classical swine fever virus for immunization of animals]. / Primenenie rekombinantnogo belka E2 virusa klassicheskoi chumy svinei dlia immunizatsii zhivotnykh.

Recombinant major surface glycoprotein E2 from virulent Shimen strain of classical swine fever virus (CSFV) has been tested for immunogenicity in animal immunization experiments. Immunization of 3-month-old piglets with 200 micrograms of recombinant protein protected the animals from lethal challenge with virulent CSFV strain. CSFV-specific antibody detection test based on competitive ELISA has been developed using the recombinant E2 protein. The test can evaluate specific antibody levels after subunit vaccination with recombinant E2 after immunization with live vaccine based on attenuated CSFV strain.

[Synthesis and immunochemical properties of the recombinant major surface glycoprotein E2 of the classical swine fever virus]. / Sintez i immunokhimicheskie svoistva rekombinantnogo glavnogo poverkhnostnogo glikoproteina E2 virusa klassicheskoi chumy svinei.

Recombinant E2 protein from vaccine strain of classical swine fever virus (CSFV) and from SCFV virulent strain Shimen was synthesized in SF-21 and High-Five cell culture with baculovirus as the expressing vector. For secretion, hydrophobic C-terminal transmembrane domain was removed and N-terminal signal polypeptide of 38 amino acids was added. Maximum accumulation of recombinant products in SF-21 cells was observed after 48 h and in medium 96 h after infection with recombinant baculovirus. In High-Five cells and in culture medium the maximum accumulation of E2 was observed after 96 h. The level of E2 expression is 5-10 micrograms/106 cells. The products of expression were purified by affinity chromatography and their specificity confirmed in immunochemical tests with a series of reference monoclonal antibodies. The product can be used for detecting antibodies to SCFV by competitive enzyme immunoassay.

[The effect of oxythiamine on tumor growth and certain biochemical and genetic characteristics of normal and tumor cells]. / Vliianie oksitiamina na opukholevyi rost i nekotorye biokhimicheskie i geneticheskie kharakteristikie normal'nykh i opukholevykh kletok.

The effect of oxythiamine (400 mg/kg) on chromosomal structure of Ehrlich ascites carcinoma cells (EAC, hyperdiploid strain) and bone marrow cells was studied in intact AF mice. The influence of the antivitamin on the rate of tumor growth was investigated in tumor-bearing mice. Oxythiamine decreased transketolase activity in hepatocytes and tumoral cells and markedly inhibited tumor growth. Amount of chromosomes was unaltered both in tumor cells and in bone marrow cells, which could be manifested as increased content of cells with impairment of chromosomal set calculated per a cell. However, the oxythiamine-induced impairment of chromosomal integrity was less distinct as compared with the effect of such mutagens as urethane and cyclophosphamide; hence, the antivitamin might be used in the courses of combined chemotherapy.

Isolation and characterization of full-length recombinant cattle PrPC protein.

Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.

[Degradation of intracellular proteins at different stages of growth of Saccharomyces cerevisiae]. / Degradatsiia vnutrikletochnykh belkov na raznykh fazakh rosta drozhzhei Saccharomyces cerevisiae.

The rate of degradation of intracellular proteins at different growth stages of Saccharomyces cerevisiae yeast was determined. It has been demonstrated that the rate of degradation of intracellular proteins increases 2--3-fold at the late exponential phase. The increase was accompanied by corresponding changes in the activities of yeast proteinases A and B. In the presence of specific yeast proteinase inhibitors (pepstatin and phenylmethylsulfonyl fluoride) the rate of protein degradation in vivo decreased. The intermediate products of cell protein degradation have been found. These TCA-insoluble products could be extracted by various solvent systems. Their subsequent brakdown was suppressed by specific proteinase inhibitors.

[Lability of the products of mitochondrial protein synthesis in Saccharomyces cerevisiae yeasts]. / Labil'nost' produktov mitokhondrial'nogo belkovogo sinteza v drozhzhakh Saccharomyces cerevisiae

Estimation of the rate of degradation of the products of mitochondrial protein synthesis in S. cerevisiae cells is reported. The method developed for this purpose is based on pulse incorporation of a labeled amino acid in the presence of an inhibitor of cytoplasmic protein synthesis and allows one to monitor postincorporation of the label. The label incorporated is shown to be rapidly released from mitochondria. Its content is decreased 2-fold during 20-30 min at the beginning and 50-60 min at the end of the exponential phase of growth. The label is detected in cytosol proteins and the TCA-soluble fraction of mitochondria, which is indicative of possible proteolysis of mitochondrial membrane proteins. Since release of the label does not undergo inhibition by specific inhibitors of yeast cell proteinases (pepstatin and phenylmethylsulfonyl fluoride), it may be assumed that these proteinases are either not involved in the digestion of the products of mitochondrial protein synthesis or do not represent a rate-limiting step of the process.

[Mitochondrial proteinase of yeast splitting the products of mitochondrial translation]. / Mitokhondrial'naia proteinaza drozhzhei, osushchestvliaiashchaia rasshcheplenie produktov mitokhondrial'noitransliatsii.

It has been shown that mitochondria of the yeast Saccharomyces cerevisiae contain proteinase, which is bound to the inner mitochondrial membrane and catalyzes the hydrolysis of mitochondrial translation products in vitro. The efficiency of proteolysis depends on the state of mitochondria: e.g. the degradation of completely formed organelles corresponding to stationary cells, is twice as low as compared to the "young" organelles typical for the beginning of a logarithmic phase of growth. The proteolysis of mitochondrial translation products can occur not only in mitochondria, but also in "inside out" submitochondrial particles. In order to prove the absence of concomitant vacuolar proteinases in preparations of mitochondria and submitochondrial particles, the specific antisera against proteinases A and B have been used. The activity of mitochondrial proteinase is completely inhibited by the natural peptide inhibitors antipain and chymostatin. Of special importance is the fact that another natural peptide inhibitor--leupeptin, having no effect on the activities of vacuolar proteinases, significantly decreases the rate of hydrolysis of mitochondrial translation products. The role of yeast mitochondrial proteinase in regulation of mitochondrial formation is discussed.

[Proteolysis of products of mitochondrial protein synthesis in isolated mitochondria of Saccharomyces cerevisiae yeasts]. / Proteoliz produktov mitokhondrial'nogo belkovogo sinteza v izolirovannykh mitokhondriiakh drozhzhei Saccharomyces cerevisiae.

Products of mitochondrial protein synthesis were specifically labeled with 3H-leucine in the presence of cycloheximide at the end of the exponential phase of yeast aerobic growth on glucose. The mitochondria isolated from these cells lost 37-40% of the label from the protein fraction during 60 min incubation at 35 degrees, which was accompanied by the accumulation of 3H-leucine in TCA-soluble fraction. This process was suppressed by phenyl-methyl sulfonyl fluoride and p-chloromercuriphenyl sulfonate, the inhibitors of proteases, and could thus be considered as the proteolysis of the products of mitochondrial protein synthesis. The proteolysis was ATP dependent and was stimulated by puromycine which is known to induce the removal of incomplete polypeptides from mitochondrial ribosomes. A body of indirect evidence allows a suggestion to be made that the observed proteolysis can hardly be due to the action of cytoplasmic proteinases.

[In vitro immunization of human lymphocytes with Epstein-Barr virus capsid antigen]. / Immunizatsiia in vitro limfotsitov cheloveka kapsidnymi antigenami virusa Epshteina-Barr.

Conditions for in vitro immunization of human lymphocytes from adult peripheral blood, tonsils and cord blood with Epstein-Barr Virus (EBV) capsid antigens have been studied. Pokeweed mitogen and B cell growth factor from Namalva cell line were shown to induce a significant production of specific antibodies by human lymphocytes stimulated with EBV. This effect made it possible to generate primary immune response in vitro using lymphocytes from EBV seronegative donors.