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Adrenoceptors as potential target for add-on immunomodulatory therapy in multiple sclerosis
No full textThis review summarizes recent findings related to the role of the sympathetic nervous system (SNS) in pathogenesis of multiple sclerosis (MS) and its commonly used experimental model – experimental autoimmune encephalomyelitis (EAE). They indicate that noradrenaline, the key end-point mediator of the SNS, acting through β-adrenoceptor, has a contributory role in the early stages of MS/EAE development. This stage is characterized by the SNS hyperactivity (increased release of noradrenaline) reflecting the net effect of different factors, such as the disease-associated inflammation, stress, vitamin D hypovitaminosis, Epstein-Barr virus infection and dysbiosis. Thus, the administration of propranolol, a non-selective β-adrenoceptor blocker, readily crossing the blood-brain barrier, to experimental rats before the autoimmune challenge and in the early (preclinical/prodromal) phase of the disease mitigates EAE severity. This phenomenon has been ascribed to the alleviation of neuroinflammation (due to attenuation of primarily microglial activation/proinflammatory functions) and the diminution of the magnitude of the primary CD4+ T-cell autoimmune response (the effect associated with impaired autoantigen uptake by antigen presenting cells and their migration into draining lymph nodes). The former is partly related to breaking of the catecholamine-dependent self-amplifying microglial feed-forward loop and the positive feedback loop between microglia and the SNS, leading to down-regulation of the SNS hyperactivity and its enhancing influence on microglial activation/proinflammatory functions and the magnitude of autoimmune response. The effects of propranolol are shown to be more prominent in male EAE animals, the phenomenon important as males (like men) are likely to develop clinically more severe disease. Thus, these findings could serve as a firm scientific background for formulation of a new sex-specific immune-intervention strategy for the early phases of MS (characterized by the SNS hyperactivity) exploiting anti-(neuro)inflammatory and immunomodulatory properties of propranolol and other relatively cheap and safe adrenergic drugs with similar therapeutic profile.Peer-reviewed manuscript: [http://intor.torlakinstitut.com/handle/123456789/663
The relationship between the cholinergic mechanism of toxicity and oxidative stress in rats during subacute diazinon poisoning
No full textDiazinon is an organophosphate pesticide (OP) that has significant potential for accidental and intentional poisoning of wildlife, domestic animals and humans. The aim of the study is to investigate the correlation between cholinesterase activity and oxidative stress parameters in liver and diaphragm by continuous monitoring as a function of time during prolonged use of diazinon. Wistar rats were treated orally with diazinon (55 mg/kg/day): 7, 14, 21 and 28 days. At the end of each period, blood, liver and diaphragm were collected to examine cholinesterase activity and enzymatic/non-enzymatic oxidative stress parameters: superoxide dismutase 1 (SOD1), catalase (CAT), thiobarbituric acid substances (TBARS), protein carbonyl groups. In all four time periods, there was a significant change in acetylcholinesterase (AChE) in erythrocytes and butyrylcholinesterase (BuChE) in blood plasma, CAT in liver and diaphragm and SOD1 in diaphragm. Parameters significantly altered during the cholinergic crisis included: cholinesterases and TBARS in liver and diaphragm and partially SOD1 in liver. Protein carbonyl groups in liver and diaphragm were significantly altered outside the cholinergic crisis. In the liver, there was a very strong negative correlation between BuChE and TBARS in all four time periods and BuChE and CAT on day 7. In the diaphragm, a very strong negative correlation was found between AChE and TBARS at days 7 and 14, and a very strong positive correlation between AChE and SOD1 at days 14, 21 and 28. A better understanding of the relationship between cholinergic overstimulation and oxidative stress may help to better assess health status in prolonged OPs intoxication
Influence of amino acid substitution on the antimicrobial activity of bacteriocin lactolisterin BU
Get PDFIntroduction: Lactolisterin BU (LBU) is a potent bacteriocin derived from Lactococcuslactis subsp. lactis
bv. diacetylactis BGBU1-4. It exhibits antimicrobial properties against Gram-positive food spoilage and
foodborne pathogens. This research aimed to explore the impact of amino acid substitution in LBU on
its antimicrobial activity by utilizing in silico prediction of LBU’ssecondary structure and amino acid substitutions.
Methods: The secondary structure of LBU was predicted using Phyre2 software. Five variants of LBU
were selected and chemically synthesized, along with unaltered LBU and BHT-B,serving as controls. Peptides were twofold diluted in distilled water, resulting in final concentrations ranging from 1000 µg/ml
to 0.5 µg/ml. An agarspot test, employing 5 µl of the dilution, was conducted on three indicatorstrains:
Lactococcus lactis BGMN1-596, Listeria monocytogenes ATCC19111, and Staphylococcus aureus
ATCC25923. The presence of inhibition zones was analyzed after overnight incubation at 37°C (S. aureus)
and 30°C (L. lactis and L. monocytogenes).
Results: Phyre2 analysis unveiled the presence of two α-helices in LBU’s structure. The majority of LBU
variants displayed altered antimicrobial activity, with some changes being genusspecific, potentially attributable to variances in cell wall composition. Some variants completely lost their activity, underscoring the significance of native amino acids or their physicochemical properties in the corresponding
positions within LBU’s structure. Furthermore, it was confirmed that chemically synthesized LBU effectively retains its antimicrobial activity.
Conclusion: Changesin amino acid composition give insight on structure-function relationship of LBU
Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants
No full textLactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25–43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1–24, the truncation at position 31 is predicted to change the structure within aa 15–31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively
Dactylis glomerata grass pollen from urban area releases more sub- pollen particles and has stronger ige response in allergic individuals than rural counterpart
Get PDFBackground and Aim: Epidemiological studies pointed at the connection betweenpollution (e.g., traffic emissions) and an increased percentage of people suffering fromrespiratory allergies, including the pediatric population. Field studies provided the mostrelevant assessment of the effects of the intensity and variety of urban and industrialcontamination on the structure and allergenic potency of pollen allergens. Therefore, theaim of the present work was to compare allergenic profiles ofDactylis glomerata pollen(DGP) collected in the specific urban and rural areas (Kruševac and suburbs), to assesspollen structures and immunoglobulin E (IgE) reactivity to pollen of school childrenpopulation allergic to grass pollens
Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome
Get PDFIntroduction: Brevibacillus laterosporus is a promising microbiological agent that can be used to prevent and control destructive diseases affecting honey bee colonies. In the presentstudy, the short-termeffect of the B. laterosporus BGSP11 bee diet on microbiota and mycobiota was investigated.Methods: The honey bee diet was supplemented with spores of B. laterosporus BGSP11 at a concentration of 1×108 CFU/mL in sucrose solution. Metabarcoding analysis of the bee microbial community profile was performed based on 16S RNA (bacteriobiota) and Internally Transcribes Spacer (ITS) region(mycobiota) obtained using MiSeq Illumina sequencing. The QIIME2 v2021.4 pipeline was used to analyze the obtained amplicon data library.Results: The results show that the BGSP11 bee diet slightly altered the bee microbiota and did not leadto potentially harmful changes in the bacterial microbiota. Moreover, it can potentially induce positivechanges, mainly reflected in the reduction of opportunistic bacteria. On the other hand, the treatmenthad a greater effect on mycobiota. However, the changesin the bee mycobiome caused by the treatmentcannot be considered a priori as beneficial or harmful,since the interaction between the bee and its mycobiome is not sufficiently studied. The observed positive changes in the bee mycobiome are mainlyreflected in the reduction of phytopathogenic fungi that may affect the organoleptic and techno-functional properties of honey.Conclusion: This pilot study suggests that the introduction of BGSP11 in beekeeping practice as a biological agent could be considered due to no harmful effects observed on the microbiota of bees
Drying without dying: revealing the role of late embryogenesis abundant proteins during desiccation in Ramonda serbica
Get PDFIntroduction: Resurrection plants (such as Ramonda serbica) can survive a long desiccation period andfully resume their metabolism upon watering. The hallmark of desiccation tolerance (DT) is the accumulation of protective, intrinsically disordered proteins(IDPs), called late embryogenesis abundant proteins (LEAPs). Although their high structural plasticity allows them to interact with various partners, nospecific cellular targets of LEAPs have been identified so far.Methods: To identify LEAPsinvolved in DT, differential transcriptome and proteome analyses of hydratedand desiccated R. serbica leaves were performed. The identified LEAPs were structurally characterisedand classified. To evaluate theirstructural propertiesin vitro and their potential functionsin vivo, the representative RsLEA proteins, were produced in Escherichia coli using recombinant DNA technology.Results: Members of the LEA4 protein family represent the majority of desiccation-inducible LEAPs. Even17 proteins belonging to the LEA4 protein family group were induced by desiccation. They show high disorder propensity (82 %), and at the same time, a high tendency to form α-helices (>80%). Although recombinant DNA technology has traditionally been used to overexpress and purify various globularproteins, the production of IDPsis challenging due to their high susceptibility to proteolytic cleavage andaggregation. Nevertheless, the representative LEAPs containing hexa-Histagsimmunoglobulin G-binding protein and a proteolytic TEV site were produced, purified and cleaved by TEV protease.Conclusion: The combination of in silico and in vitro results will be crucial for the identification of endogenous partners of LEAPs, providing further insight into their role in DT
Highly stable and versatile α-amylase from Anoxybacillus vranjensis ST4 suitable for various applications
No full textα-Amylase from the thermophilic bacterial strain Anoxybacillus vranjensis ST4 (AVA) was cloned into the pMALc5HisEk expression vector and successfully expressed and purified from the Escherichia coli ER2523 host strain. AVA belongs to the GH13_5 subfamily of glycoside hydrolases and has 7 conserved sequence regions (CSRs) distributed in three distinct domains (A, B, C). In addition, there is a starch binding domain (SBD) from the CBM20 family of carbohydrate binding modules (CBMs). AVA is a monomer of 66 kDa that achieves maximum activity at 60–80 °C and is active and stable over a wide pH range (4.0–9.0). AVA retained 50 % of its activity after 31 h of incubation at 60 °C and was resistant to a large number of denaturing agents. It hydrolyzed starch granules very efficiently, releasing maltose, maltotriose and maltopentaose as the main products. The hydrolysis rates of raw corn, wheat, horseradish, and potato starch, at a concentration of 10 %, were 87.8, 85.9, 93.0, and 58 %, respectively, at pH 8.5 over a 3 h period. This study showed that the high level of expression as well as the properties of this highly stable and versatile enzyme show all the prerequisites for successful application in industry
Improvement of nutritional and bioactive properties of barley b-glucan-based food products using Bacillus subtilis 168 endo-b-1,3-1,4-glucanase
No full textThe combination of b-oligosaccharides from enzymatically hydrolysed barley b-glucan has attracted interest recently due to its positive effects on human health. This study aimed to assess the impact of the
endo-b-1,3-1,4-glucanase enzyme from Bacillus subtilis 168 on improving the nutritional and bioactive
properties of barley b-glucan. A new procedure for the isolation of b-glucan was developed, at a lower
temperature (45 °C), enabling purity from starch contamination, without affecting the yield (6 g b-glucan
from 100 g of barley flour). The endo-b-1,3-1,4-glucanase is cloned into E. coli pQE_Ek enables the high
production and purification (82% yield, 1.8 mg mL 1 and 440 U mg 1
) of an enzyme identical to the
natural one (25.5 kDa). The enzymatic reaction showed high efficiency of b-glucan degradation by recombinant enzyme, giving a mixture of products (of which 3-O-b-cellobiosyl-D-glucose and 3-O-b-cellotriosylD-glucose are the most abundant), the reduction of viscosity (17%) and increase in antioxidant capacities
by 15.2%, 30.9% and 44.0% assessed by ABTS, DPPH and ORAC, respectively. These results indicate
the possible application of endo-b-1,3-1,4-glucanase enzyme in improving the properties of barley bglucan used as functional foods
Neurotoxic effect of Vipera ammodytes venom on the rat isolated diaphragm model
Get PDFPoskok (Vipera ammodytes) predstavlja najzastupljeniju zmiju otrovnicu u Srbiji i na Balkanu, kako po brojnosti tako i po arealu rasprostranjenosti. U poređenju sa venomima ostalih otrovnica iz familije Viperidae na Balkanu, toksičnost venoma poskoka je najveća. Neurotoksičnost sirovog venoma poskoka smo ispitivali u in vitro uslovima na osnovu kontrakcija preparata dijafragme izazvanih poljnom električnom stimulacijom i aktivnosti enzima: acetilholinesteraze (AChE) i ukupnih ATP-aza u dijafragmi.Formirano je 5 grupa pacova (n=5): kontrola (dijafragma bez venoma), dijafragma sa venomom, dijafragma sa smešom venom/antivenom („Viekvin“, Torlak, Srbija) u odnosima 1:2, 1:10 i 1:20 (m/m). Kontrakcije dijafragme su opale na 50% kontrolnih kontrakcija: pod uticajem venoma za 62,00±2,31minuta, a sa smešom venom/antivenom 1:2 za 78,50±7,51 minuta, 1:10 za 150,00±17,32 minuta, 1:20 za 307,50±2,89 minuta. Statistička razlika (p0,05) pod dejstvom venoma. Venom inhibira aktivnost ukupnih ATP-aza dijafragme za 51,81% u odnosu na kontrolu. Venom poskoka ne deluje na AChE i nikotinske receptore u neuro-mišićnoj sinapsi dijafragme, već narušava energetski metabolizam mitohondrija.Vipera ammodytes is the most common venomous snake in Serbia and the Balkans, both in terms of numbers and area of distribution. Compared to the venoms of other poisonous snakes from the family Viperidae in the Balkans, the toxicity of the V. ammodytes venom is the highest. We investigated the neurotoxicity of crude venom in vitro using contractions of the diaphragm preparation induced by electric field stimulation and enzyme activity: acetylcholinesterase (AChE) and total ATPases in the diaphragm. Five groups of rats were formed (n=5): control (diaphragm without venom), diaphragmwith venom, diaphragm with mixture of venom/antivenom ("Viekvin", Torlak, Serbia) in the ratio 1:2, 1:10 and 1:20 (m/m). Diaphragmatic contractions decreased to 50% of control contractions: under the influence of venom for 62.00±2.31 minutes, and with venom/antivenom mixture 1:2 for 78.50±7.51 minutes, 1:10 for 150.00±17.32 minutes, 1:20 for 307.50±2.89 minutes. A statistical difference (p 0.05) under the influence of the venom. Venom inhibits the total ATPases activity of the diaphragm by 51.81% compared with control.The venom of V. ammodytes does not act on AChE and nicotinic receptors in the neuromuscular synapse of the diaphragm, but interferes with mitochondrial energy metabolism.Abstract boo