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The Nobel Prize in Chemistry 2023

The Nobel Prize in Physiology or Medicine 2023

Contents UBJ, 2023, Volume 95, Issue 5

Isolation and characterization of Bacillus sp. IMV B-7883 proteases

O. V. Gudzenko*, L. D. Varbanets

Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv, Ukraine;

Received: 20 July 2023; Revised: 12 September 2023;
Accepted: 27 October 2023; Available on-line: 06 November 2023

The representatives of Bacillus are some of the best protease producers studied so far since they exhibit broad substrate specificity, significant activity, stability, simple downstream purification, short period of fermentation and low cost. Earlier, we showed that Bacillus sp. IMV B-7883 strain synthesizes an extracellular proteases, which exhibit elastolytic and fibrinogenolytic activity. The aim of the work was to isolate and purify these enzymes from the culture liquid of the Bacillus sp. IMV B-7883 strain, as well as to study their properties. Isolation and purification of proteases was carried out by precipitation of the culture liquid with ammonium sulfate, gel permeation and ion exchange chromatography and rechromatography on Sepharose 6B. As a result, proteases with elastolytic and fibrinogenolytic activity with a molecular weight of 23 and 20 kDa respectively were isolated with elastase activity increased by 63.6 and fibrinogenolytic activity by 44.1 times. The enzyme with elastase activity had a pH-optimum of 7.0 and hydrolyzed only elastin, while the enzyme with fibrinogenolytic activity was an alkaline protease with a pH-optimum of 8.0 and in addition to fibrinogen, showed specificity for fibrin and, in trace amounts, for collagen.

Gamma-aminobutyric acid modulates antioxidant and osmoprotective systems in seedlings of Triticum aestivum cultivars differing in drought tolerance

Yu. E. Kolupaev1,2,3*, I. V. Shakhov1,3, A. I. Kokorev1,
L. Kryvoruchko2, T. O. Yastreb1,4

1Yuriev Plant Production Institute, National Academy of Agrarian Sciences of Ukraine, Kharkiv;
2Poltava State Agrarian University, Poltava, Ukraine;
3State Biotechnological University, Kharkiv, Ukraine;
4Crop Research Institute, Prague, Czech Republic

Received: 28 August 2023; Revised: 30 September 2023;
Accepted: 27 October 2023; Available on-line: 06 November 2023

The stress-protective effects of plant neurotransmitters, including gamma-aminobutyric acid (GABA) have been intensively examined in recent years. However, studies on the GABA influence on stress protective systems in bread wheat cultivars with different drought adaptation strategies are still lacking. The aim of this work was to estimate the GABA effect on the state of antioxidant and osmoprotective systems in etiolated seedlings of two wheat cultivars differing significantly in drought tolerance, namely Doskonala (non-drought-resistant) and Tobak (drought-resistant) under model drought induced by PEG 6000. Two-day-old seedlings were transferred to 15% PEG 6000 and incubated for two days in the absence or presence of GABA. Treatment with 0.1 and 0.5 mM GABA significantly reduced the growth-inhibitory effect of PEG 6000 on the roots and shoots of both cultivars, but to a greater extent on those of non-resistant Doskonala. It was shown that GABA treatment reduced drought-induced accumulation of H2O2 and MDA, stabilized SOD and GPX activity, the level of sugars, anthocyanins and flavonoids in seedlings of both cultivars. Meanwhile GABA treatment enhanced the stress-induced increase in proline content in the Doskonala cultivar, but decreased it in the Tobak, completely prevented stress induced decrease in anthocyanins and flavonoids level in the Tobak and only partially in Doskonala cultivar. Thus, the stabilization of the stress-protective systems functioning in the wheat cultivars and variety-dependent differences in response to GABA were revealed.

Prevalence of 4a/4b polymorphic variants of the eNOS gene introne in patients with different types of encephalopathies

K. V. Duve

I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine;

Received: 26 September 2023; Revised: 26 October 2023;
Accepted: 27 October 2023; Available on-line: 06 November 2023

Questions related to the disruption of vasoregulatory processes as essential factor in neurological pathology require further study. The leading role in the vasoregulatory mechanisms is played by endothelial NO synthase which gene has 15 allelic variants. Recent data indicated a probable association between eNOS gene polymorphism and cerebrovascular diseases. The aim of the present work was to study the prevalence of the 4a/4b introne polymorphism of the eNOS gene in patients with various types of encephalopathies and to evaluate the influence of a particular genotype of the studied gene on the occurrence and/or progression of encephalopathy. A total of 96 patients with encephalopathies of various genesis: chronic traumatic encephalopathy, chronic alcohol-induced encephalopathy, chronic vascular encephalopathy, post-infectious encephalopathy were involved in the study. The patients received inpatient treatment in the neurological department of “Ternopil Regional Clinical Psychoneurological Hospital”. Molecular and genetic differentiation of the studied gene variants was carried out by allele-specific PCR or PCR-restriction fragment length polymorphism analysis by standard operational protocols. Analysis of the frequency distribution of eNOS gene 4a/4b polymorphic variant showed that 4b allele prevailed among patients with all types of encephalopathies carriers­. Relative to practically healthy individuals the difference was found only in patients with chronic vascular encephalopathy (CVE), among whom about 39% were carriers of the 4a allele. The presence of the 4a allele was shown to increased the risk of CVE occurrence and/or progression by 4.5 times. The results obtained suggest the reasonability to include the 4a/b intron polymorphism of the eNOS gene in a genetic panel to monitor patients CVE.

C(60) fullerene attenuates the signs of acute renal failure in rats under rhabdomyolysis due to inhibition of oxidative stress

O. Omelchuk1, S. Prylutska2, D. Nozdrenko3,
O. Motuziuk1,3, I. Vareniuk3, K. Bogutska3,
O. Vygovska4, А. Zholos3, Yu. Prylutskyy3*

1Lesya Ukrainka Volyn National University, Lutsk, Ukraine;
2National University of Life and Environmental Science of Ukraine, Kyiv, Ukraine;
3Taras Shevchenko National University of Kyiv, Ukraine;
4Bogomolets National Medical University, Kyiv, Ukraine

Received: 14 August 2023; Revised: 18 September 2023;
Accepted: 27 October 2023; Available on-line: 06 November 2023

Rhabdomyolysis, as an acute stage of myopathy is known to be associated with the accumulation of muscle breakdown products, acute renal failure and oxidative stress. The goal of the study was to evaluate the effect of C60 fullerene as an antioxidant on kidney damage in the model of glycerol-induced rhabdomyolysis in rats. The study was conducted on male Wistar rats, divided into the following experimental groups: control animals, animals intramuscularly injected with glycerol in a doses of 5, 10 and 15 mg/kg and those intraperitoneally injected daily with C60 fullerene aqueous solution (C60FAS) in a dose of 1 or 2 mg/kg at 48 h after glycerol administration­. Monitoring of the biochemical and morphological indicators was carried out on 3rd, 6th and 9th days of the experiment. A close correlation between the acute renal damage severity, increased creatinine and urea level, superoxide dismutase (SOD) and catalase (CAT) activity in the blood of rats was observed. It was shown that in rats which received 2 mg/kg of C60FAS the renal glomeruli size and necrosis manifestations were attenuated, whereas SOD and CAT activity in the blood was significantly decreased. The results obtained may be useful for developing approaches to the treatment of pathological conditions of the muscular system caused by rhabdomyolysis and associated oxidative stress.

Bioenergetic functions of mitochondria in liver, pancreatic acinar cells, and sperm cells of rats fed short-term high-fat or high-fat high-sugar diets

B. V. Manko1*, N. M. Kozopas1,2, H. M. Mazur1,
A. М. Voityk1, B. O. Manko1, V. V. Manko1

1Ivan Franko National University of Lviv,
Department of Human and Animal Physiology, Lviv, Ukraine;
2Danylo Halytsky Lviv National Medical University,
Department of Clinical Laboratory Diagnostics, Lviv, Ukraine;

Received: 26 September 2023; Revised: 23 October 2023;
Accepted: 27 October 2023; Available on-line: November 2023

An unhealthy diet often is a cause of obesity, chronic inflammation, and metabolic disruption in multiple organs. However, the direct influence of elevated lipid or sugar consumption on liver, pancreatic, and sperm mitochondria is not well understood. The aim of the study was to investigate the functional activity of mitochondria of liver, pancreatic acinar cells, and sperm cells in rats on a short-term (7 weeks) diet with high fat or high fat and high sugar content. Male Wistar rats were on a basic, high-fat or high-fat high-sugar diet for 7 weeks. At the end of the experiment, visceral fat mass, blood glucose and lipids were measured. Mitochondrial functional activity was evaluated with oxygen consumption assay. In isolated pancreatic acinar cells, NAD(P)H autofluorescence and mitochondrial membrane potential were also studied. No difference in body mass was observed between the 3 groups at the end of the experiment. Visceral fat mass was slightly but significantly elevated in rats on a high-fat high-sugar diet. Both diets did not affect plasma glucose or triglyceride levels but caused a modest elevation of total plasma cholesterol. Respiration and oxidative phosphorylation of isolated liver mitochondria were not affected by any experimental diet. In pancreatic acinar cells, a high-fat diet caused a significant decrease of basal respiration by ~15%, but no effects were observed on the maximal rate of uncoupled respiration, mitochondrial membrane potential, or NAD(P)H autofluorescence. In these cells, a ketone body 3-hydroxybutyrate caused elevation of uncoupled respiration and NAD(P)H level irrespectively of the diet. Diets did not cause any change in sperm concentration, viability or motility. Surprisingly, in animals on a high-fat high-sugar diet, a significant increase in both basal and maximal respiration of sperm cells was observed. Collectively, these data show that while the elevated fat and sugar content in the diet does not cause significant obesity, no detrimental effects on mitochondria of the liver, pancreas, and sperm cells are observed.

Novel biochemical markers for the prediction of renal injury in beta-thalassemia major pediatric patients

A. J. Shwayel1*, A. M. Jewad1, M. Q. Abdulsattar2

1Department of Medical Laboratory Technology, Health and Medical Technical College,
Southern Technical University, Basrah, Iraq;
2Thi-Qar Health Directorate, Iraq;

Received: 07 June 2023; Revised: 14 August 2023;
Accepted: 27 October 2023; Available on-line: 06 November 2023

Beta-thalassemia major is a severe inherited disorder characterized by inadequate production of hemoglobin beta chains, ineffective erythropoiesis, chronic hemolysis and necessitates lifelong transfusions, which lead to iron overload. The disease manifests itself in early childhood and persists throughout an individual’s life with a high risk of developing renal impairment, which cannot be reliably determined using routine markers. The objective of this research was to apply biomarkers to the evaluation of renal injury in pediatric­ patients within the age range of 1–14 years diagnosed with beta-thalassemia major. In the case-control study, the blood samples obtained in the Genetic Hematology Center in Thi-Qar Iraq Province were used: 60 samples from healthy individuals and 60 samples from the patients with beta-thalassemia, subdivided into 1-7 and 8-14 years old groups. The levels of hemoglobin, ferritin, creatinine and potassium were estimated with standard tests, enzyme-linked immunoassay was used to determine the level of neutrophil gelatinase-associated lipocalin (NGAL) and beta-2-microglobulin (β2M) as novel markers of tubular and glomerular dysfunctions. The study revealed a statistically significant decrease in hemoglobin, serum potassium levels and an increase in ferritin, NGAL and β2M levels in the patients from both groups compared to controls and elevation of creatinine level in the 8–14-year-old group. It was concluded that NGAL and β2M levels may be considered indicators for the early diagnosis of renal injury in pediatric patients with beta-thalassemia, as these biomarkers exhibit elevated levels before an increase in creatinine is observed.

Assessment of urinary podocalyxin as a biomarker of early diagnosis of hypertensive nephropathy

I. Kostovska*, K. Tosheska-Trajkovska, D. Labudovic,
S. Cekovska, O. Kostovski, G. Spasovski

Faculty of Medicine, Ss. Cyril and Methodius University in Skopje, North Macedonia;

Received: 31 August 2023; Revised: 28 September 2023;
Accepted: 27 October 2023; Available on-line: 06 November 2023

Chronically high blood pressure-related kidney injury is known as hypertensive nephropathy (HN). Podocyte damage in the pathogenesis of this disease can result in the release of the sialoglycoprotein podocalyxin into the urine, so podocalyxin may be useful in the early diagnosis of HN. The purpose of the study was to examine the relationships between urine podocalyxin level and clinical and biochemical parameters in individuals with HN and to assess the diagnostic utility of urinary podocalyxin as an early marker of HN. Participants (114 individuals) were enrolled in this cross-sectional study, including 30 healthy controls and 84 patients with clinically proven chronic hypertension (CH). Biochemical tests were performed on the blood samples. Urinary microalbumin and creatinine levels were measured using immunoturbidimetric and spectrophotometric methods, respectively; urinary podocalyxin level was estimated with ELISA. All CH patients were classified into subgroups according to urine microalbumin/creatinine ratio (UM/CR) and the stage of chronic kidney disease (CKD). The results obtained showed that urinary podocalyxin level was significantly increased in both UM/CR and CKD staging subgroups compared with the healthy control group. A gradual increase in urinary podocalyxin level with CKD stage, especially in IV and V stages, and the higher sensitivi­ty of urinary podocalyxin as compared to UM/CR ratio in early detection of HN was demonstrated. It was concluded that urinary podocalyxin may be an important and highly sensitive marker for early diagnosis of hypertensive nephropathy in patients with chronic hypertension.