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Different mice inbred strains humoral immune response against human prostate-specific antigen

O. Yu. Galkin1,2, A. G. Komar3, O. B. Besarab1

1National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”;
e-mail: alexfbt@gmail.com;
2Propharma Plant Ltd., Kyiv;
3Ukrainian Medical Center of Certification of Ministry of Health of Ukraine, Kyiv

Received: 21 August 2018; Accepted: 13 December 2018

The aim of the study was an investigation of humoral immune response to prostate-specific antigen (PSA) of different mice inbred strains for further development of recommendations for appropriate immunization schemes for monoclonal antibodies (McAbs) obtaining. The study was conducted using: Balb/c and NZB mice; PSA from human sperm (as immunogen). In case of booster immunization immunogen was previously diluted to the desired concentration (10 or 30 µg per 100 µl) in saline, and injected in the tail vein or intraperitoneally. In case of other immunizations, we prepared emulsion solution of immunogen with adjuvant to final concentration of 10 or 30 µg per 100 µl: PSA was dissolved in saline, the same volume of adjuvant was added, and mixture was thoroughly mixed to form a stable emulsion. When subcutaneous administration, total dose of 100 µl was divided into two equal parts and injected in the hind paw of mice. Intraperitoneal immunization was performed by single administration of 100 µl of emulsion. The level of specific antibodies was determined by titration of blood sera from animals in indirect ELISA. Series of experiments were conducted to determine the level of humoral response of mice of different inbred strains (Balb/c and NZB) for multi-stage immunization with different duration with different amounts of PSA (10 and 30 μg), with different immunoglobulin administration (intraperitoneal and subcutaneously), and with different adjuvants (Freund’s complete and incomplete adjuvants, FCA/FIA). Final booster immunization at a dose of 30 μg was performed either in the same manner as the previous administration of the immunogen, or intravenously in the physio­logical saline solution.  Dependencies of the humoral immune response of Balb/c and NZB mice against PSA on the route of administration of immunogen, the dose and duration of immunization were established. It was shown that intraperitoneal administration provided formation of higher titers of specific antibodies in case of both mice strains. Balb/c mice lines more rapidly responded to PSA, than an NZB mice (for all investigated schemes immunization). It was shown that the most effective immunization scheme was three times intraperitoneal administration with 10 µg of PSA for 8 weeks (the first immunization with FCA, and the rest – with FIA) and booster immunogen intravenous administration in saline solution.

Altered sirtuins 1 and 2 expression in the brain of rats induced by experimental diabetes and the ways of its correction

M. M. Guzyk1, T. M. Tykhonenko1, K. O. Dyakun1,
L. V. Yanitska2, I. B. Pryvrotska3, T. M. Kuchmerovska1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Bogomolets National Medical University, Kyiv, Ukraine;
3I. Horbachevsky Ternopil State Medical University, Ukraine;
e-mail: tkuchmerovska@gmail.com

Received: 03 August 2018; Accepted: 13 December 2018

The molecular pathogenesis of diabetic encephalopathy (DE), one of the serious complications of diabetes mellitus, is complex. In this study, we examined whether expression levels of SIRT1 and SIRT2 were the key for the development of brain dysfunctions and whether PARP-1 inhibitors could affect the expression of these proteins for prevention the development of DE in rats with type 1 diabetes. After 10 weeks of the streptozotocin-induced diabetes mellitus (70 mg/kg), Wistar male rats were treated by i.p. injection with PARP-1 inhibitors, 1.5-isoquinolinediol (ISO) or nicotinamide (NAm) (3 or 100 mg/kg/daily i.p., respectively) for 2 weeks. The rats with blood glucose levels over 19.7 ± 2.1 mmol/l were taken into experiments. Western blots were performed to evaluate effects of PAPR-1 inhibitors on the levels of sirtuins, SIRT1 and SIRT2 expression. Diabetes induced significant reduction of SIRT1 expression and SIRT2 overexpression in brain nuclear extracts of diabetic rats compared to non-diabetic control. In brain, NAm attenuated SIRT2 overexpression in nuclear extracts of diabetic rats and slightly elevated SIRT1 expression, while ISO didn’t affect expression of both sirtuins in diabetic rats. Furthermore, it was observed that in brain of diabetic rats, the ratio of free NAD/NADH couples decreased 3.1-fold compared to non-diabetic control. The administration of ISO increased only slightly the ratio of free NAD/NADH couples in the brain of diabetic rats while NAm increased this parameter 1.7-fold compared to diabetic rats. Therefore, we concluded that alterations in the expression of SIRT1 and SIRT2 in brain cell nuclei of diabetic rats can lead to the development of brain dysfunctions. One of the neuroprotective mechanisms of NAm action can also be realized through inhibition of SIRT2 expression in brain cell nuclei that down-regulate progression of diabetes-induced alterations and can be a therapeutic option for treatment of brain dysfunctions.

Рantoea agglomerans lipopolysaccharides: structure, functional and biological activity

L. D. Varbanets, Т. V. Bulyhina, L. А. Pasichnyk, N. V. Zhytkevich

Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
e-mail: varbanets_imv@ukr.net

Received: 02 September 2018; Accepted: 13 December 2018

This review analyzed literature data, as well as our own research on lipopolysaccharides (LPS) of gram-negative bacteria, focusing mainly on Pantoea agglomerans, a member of the Enterobacteriaceae fami­ly. The unique structures of O-specific polysaccharide chains of LPS from Pantoea agglomerans represented by both linear and branched tetra- and pentasaccharide repeating units were described for the first time. The heterogeneity of the LPS molecule itself and the presence of several LPS in the bacterial cell, which differ in the structure of lipids A, O-specific polysaccharide chains, serological activity, as well as endotoxic properties, such as toxicity and pyrogenicity, were shown. Such heterogeneity represents one of the mechanisms of LPS multifunctionality. Based on the antigenicity of LPS, serotyping of P. agglomerans strains and their assignment to 10 serogroups were carried out for the first time. The high immunomodulatory activity of P. agglomerans LPS suggests the possibility to use their oligosaccharide fragments in the development of conjugated vaccines against diseases caused by gram-negative bacteria.

Dmytro Oleksiyovych Melnychuk

On the 75th anniversary of birthday

Nobel Laureates of the early 20th century E. Behring, I. Mechnikov, P. Ehrlich, C. Richet, J. Bordet, K. Landsteiner and their contribution to the development of molecular immunology

V. M. Danilova, R. P. Vynogradova, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: valdan@biochem.kiev.ua

The discoveries of immunologists have often been recognized as the most significant in the field of medicine and physiology, since the immune system is extremely vital for the organism, and the study of the principles of its functioning is of fundamental importance to the prevention (vaccination), diagnosis and therapy of many diseases. This article refers to the scientists of the early twentieth century, who received the most prestigious scientific award – the Nobel Prize in Physiology or Medicine and who built the groundwork for the development of immunology as a science. Thus, in 1901, E. von Behring received the first Nobel Prize “for his work on serum thera­py, especially its application against diphtheria, by which he has opened a new road in the domain of medical science and thereby placed in the hands of the physician a victorious weapon against illness and deaths”; in 1908, I. Mechnikov and P. Ehrlich received the Nobel Prize in Physiology or Medicine for the creating of the cellular and humoral theory of immunity; in 1913 – C. Richet – “in reco­gnition of his work on anaphylaxis”; in 1919 – J. Bordet – “for his discoveries relating to immunity (the role of complement, mechanisms of precipitation, agglutination…)”; in 1930 – K. Landsteiner – “for his discove­ry of human blood groups”. Their works spurred the development of modern molecular immunology – the science of the organization and function of the immune system, as an effective defense barrier in the living organism, which recognize and distinguish between “self” and “non-self”.

Long-term hypocholesterolemic effect of amidated alginate in rats

M. Marounek1, Z. Volek1, T. Taubner1, D. Dušková1, L. Kalachniuk2

1Institute of Animal Science, Prague, Czech Republic;
e-mail: marounek.milan@vuzv.cz;
2National University of Life and Environmental Sciences of Ukraine, Kyiv;
e-mail: kalachnyuk_liliya@nubip.edu.ua; lilkalachnyuk@gmail.com

The effect of octadecylamide of alginic acid on blood serum and hepatic cholesterol, and the faecal output of fat and sterols was examined in female rats fed diets containing cholesterol and palm fat at 10 and 50 g/kg, respectively for 10 weeks. Amidated alginate, supplied at 10 and 20 g/kg, significantly decreased serum cholesterol from 5.25 to 2.99 and 2.39 µmol/ml, respectively, and decreased hepatic cholesterol from 30.7 to 12.3 and 9.4 µmol/g, respectively. Amidated alginate increased the faecal output of fat and at higher dosing significantly decreased faecal output of bile acids. Faecal output of bile acids and hepatic cholesterol significantly correlated (r = 0.791; P < 0.001). The results of the present experiment showed that hypocholesterolemic effect of amidated alginate persisted within 10 weeks of feeding.

Preventive effect of N-stearoylethanolamine on memory disorders, blood and brain biochemical parameters in rats with experimental scopolamine-induced cognitive impairment

T. M. Horid’ko1, H. V. Kosiakova1, A. G. Berdyshev1, O. F. Meged1,
O. V. Onopchenko1, V. M. Klimashevsky1, О. S. Tkachenko1, V. R. Bazylianska1,
V. O. Kholin2, K. O. Peschana2, S. A. Mykhalskiy2, N. M. Hula1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of Gerontology, National Academy of Medical Sciences of Ukraine, Kyiv;
e-mail: TanGoRi@ukr.net

The impairment of cognitive functions is the most studied medical and social problem nowadays. The aim of this study was to evaluate the protective effects of N-stearoylethanolamine (NSE) on memory state, blood and brain biochemical parameters in rats under scopolamine-induced cognitive impairment. The results of this study shown that NSE administration to rats per os (5 mg/kg, 5 days, during last 3 days NSE was administrated 20 min prior to scopolamine injection (1 mg/kg, once daily for 3 days, intraperitoneally)) prevented the development of memory impairment. In particular, NSE action was associated with the prevention of increase in acetylcholinesterase activity, changes in phospholipid, free and esterified cholesterol level in hippocampus and frontal cortex, and disruption in pro-/antioxidant balance in blood and studied brain sections. Considering the above mentioned biological effects, NSE is a promising drug candidate for integrative therapy of cognitive impairment of different profiles.

The effects of PDK4 inhibition on AMPK protein levels and PGC-1α gene expression following endurance training in skeletal muscle of Wistar rats

S. Aminizadeh1, Y. Masoumi-Ardakani1, B. Shahouzehi2

1Physiology Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Iran;
e-mail: soheilaminizadeh@gmail.com; ymab125@yahoo.com;
2Cardiovascular Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Iran;
e-mail: bshahouzehi@gmail.com

There are regulatory networks in cells which surveil the physiological and environmental states. These cellular regulations are conducted through gene expression modulation. Skeletal muscle is able to adapt shortly and produce ATP at different conditions. AMPK (AMP-activated protein kinase) and PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator-1alpha) are important regulators of cellular energy homeostasis. We designed this study to examine the effects of interactions between endurance training­ and PDK4 (pyruvate dehydrogenase kinase 4) inhibition on AMPK and PGC-1α expression in rat skeletal muscle. Thirty-two male Wistar rats were randomly selected and divided into 4 groups (n = 8); Group 1 control which did not receive any treatment, Group 2 received dichloroacetic acid (DCA) (150 mg/kg/day), Group 3 (endurance training group), Group 4 which received DCA and performed endurance training. AMPK protein expression, PDK4 and PGC-1α gene expression were measured by western blotting and real-time PCR, respectively. Our data showed that PDK4 inhibition caused AMPK protein elevation. Endurance training­ (group 2) and PDK4 inhibition (group 4) induce significant enhancement of PGC-1α gene expression compared to control group. The group which received DCA showed significant elevation of PDK4 gene expression compared to control group (P = 0.001), also other two groups (groups 2 & 3) showed significant elevation of PDK4 gene expression compared to control (P = 0.006). It seems that the combination of endurance training and PDK4 inhibition by up-regulation of PGC-1α expression, effectively improves energy state and performance in skeletal muscle.

Disturbance of the transmembrane phosphatidylserine asymmetry in hepatocytes as an apoptosis marker under the action of xenobiotics on rats

O. A. Nakonechna, L. A. Babijchuk, A. I. Bezrodna

Kharkiv National Medical University, Kharkiv, Ukraine;
e-mail: bezrodnaya.ai@gmail.com

It has been reported that unfavorable chemical environmental factors affect the functional state of liver, activate free radical processes against the background of the reduced antioxidant activity, change physico-chemical properties and membrane phospholipid composition of  hepatocytes. The aim of our research was to estimate phosphatidylserine distribution in the phospholipid bilayer of hepatocyte membranes and apoptosis stages in hepatocytes of rats under the influence of surfactants: ethyleneglycol (EG), polyethyleneglycol 400, (PEG-400) and polypropyleneglycol (PPG) at a dose of 1/10 DL50. It was found in the  subacute toxicological experiment on rats that the investigated xenobiotics EG, PEG-400 and PPG at a dose of 1/10 DL50 caused phosphatidylserine translocation to the outer membrane in the phospholipid bilayer of hepatocytes. This is a specific signal for macrophages aiming at recognition and elimination of apoptotic cells. Analysis of cell death modes under the influence of the investigated xenobiotics at a dose of 1/10 DL50 revealed that the intake of xenobiotics was associated with  an increase in the amount of apoptotic/necrotic hepatocytes.