Modeling and Optimization of ß-Galactosidase Entrapping in Polydimethylsiloxane-Modified Silica Composites.

Protein entrapment has multiple applications in enzymatic hydrolysis, drug delivery, etc. Here, we report the studies that successfully utilized the Box-Behnken design to model and optimize the parameters of ß-galactosidase entrapment in sol-gel-derived silica composites. We have also demonstrated the influence of polymer-polydimethylsiloxane as a composite modifying agent on the activity of entrapped enzymes. We have determined how different sol-gel process parameters influence the activity of entrapped enzymes. The highest impact on ß-galactosidase activity was exerted by the water:tetramethoxysilane ratio, followed by polydimethylsiloxane content. Optimized synthesis parameters have been utilized to obtain a composite with maximum ß-galactosidase activity. Performed porosity studies have shown that the addition of polydimethylsiloxane increased the pore diameter. Microscopy studies demonstrated that polydimethylsiloxane-modified composites are softer and less rough. Studies of ß-galactosidase activity using the o-NPG test showed statistically significant shifts in the enzyme temperature and pH profiles compared to the soluble form. An improvement in the reusability of the enzyme and a significant increase in the thermal stability was also observed. When lactose was used, a strong correlation was observed between the substrate concentration and the type of the catalyzed reaction. Moreover, we have demonstrated that the yields and rates of both lactose hydrolysis and galactooligosaccharides formation were correlated with reaction temperature and with the presence of polydimethylsiloxane. All these findings provide the opportunity for industrial use of optimized PDMS-modified silica composites in lactose elimination from dairy products, e.g., milk or whey.

GSH Protects the Escherichia coli Cells from High Concentrations of Thymoquinone.

The aim of the present study was to evaluate the potential protective effect of glutathione (GSH) on Escherichia coli cells grown in a high concentration of thymoquinone (TQ). This quinone, as the main active compound of Nigella sativa seed oil, exhibits a wide range of biological activities. At low concentrations, it acts as an antioxidant, and at high concentrations, an antimicrobial agent. Therefore, any interactions between thymoquinone and glutathione are crucial for cellular defense against oxidative stress. In this study, we found that GSH can conjugate with thymoquinone and its derivatives in vitro, and only fivefold excess of GSH was sufficient to completely deplete TQ and its derivatives. We also carried out studies on cultures of GSH-deficient Escherichia coli strains grown on a minimal medium in the presence of different concentrations of TQ. The strains harboring mutations in gene ΔgshA and ΔgshB were about two- and fourfold more sensitive (256 and 128 µg/mL, respectively) than the wild type. It was also revealed that TQ concentration has an influence on reactive oxygen species (ROS) production in E. coli strains-at the same thymoquinone concentration, the level of ROS was higher in GSH-deficient E. coli strains than in wild type.

Influence of Supercritical Carbon Dioxide Extraction Conditions on Extraction Yield and Composition of Nigella sativa L. Seed Oil-Modelling, Optimization and Extraction Kinetics regarding Fatty Acid and Thymoquinone Content.

Nigella sativa L. is cultivated in many regions and its seeds have found use in variety of foods, but also in traditional medicine due to high content of biologically active essential oils. In this work optimization of supercritical carbon dioxide extraction from N. sativa seeds was performed using response surface methodology to describe the influence of extraction conditions on oil yield. Kinetics of oil and thymoquinone extraction were analyzed as well. It was demonstrated that in order to collect thymoquinone-rich N. sativa oil fraction, appropriate for health-related applications, the extraction should be carried out at 40 °C and 10-15 MPa. Following application of higher pressure of 35 MPa enables effective extraction of remaining oil rich in polyunsaturated fatty acids suitable for use in food industry. Thymoquinone-dependent antibacterial activity of the N. sativa seed oil was observed against bacterial pathogens: Haemophilus influenzae, Staphylococcus haemolyticus, Staphylococcus epidermidis, Enterococcus faecalis and Escherichia coli.

Evidence for interactions between homocysteine and genistein: insights into stroke risk and potential treatment.

Elevated plasma homocysteine (2-amino-4-sulfanylbutanoic acid) level is a risk factor for stroke. Moreover, it has been suggested that high levels of homocysteine in the acute phase of an ischemic stroke can predict mortality, especially in stroke patients with the large-vessel atherosclerosis subtype. In clinical studies, supplementation with genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) decreased plasma homocysteine levels considerably. Therefore, genistein could be considered as a potential drug for prevention and/or treatment of stroke. However, the mechanism of the effect of genistein on homocysteine level remains to be elucidated. In this report, direct functional interactions between homocysteine and genistein are demonstrated in in vitro experimental systems for determination of methylenetetrahydrofolate reductase (MetF) and glutathione peroxidase (GPx) activities, reconstructed with purified compounds, and in a simple in vivo system, based on measurement of growth rate of Vibrio harveyi and Bacillus subtilis cultures. Results of molecular modelling indicated that homocysteine can directly interact with genistein. Therefore, genistein-mediated decrease in plasma levels of homocysteine, and alleviation of biochemical and physiological effects of one of these compounds by another, might be ascribed to formation of homocysteine-genistein complexes in which biological activities of these molecules are abolished or alleviated.

The model homologue of the partially defective human 5,10-methylenetetrahydrofolate reductase, considered as a risk factor for stroke due to increased homocysteine level, can be protected and reactivated by heat shock proteins.

The A222 V substitution in the human MTHFR gene product (5,10-methylenetetrahydrofolate reductase) is responsible for a decreased activity of this enzyme. This may cause an increased homocysteine level, considered as a risk factor for arteriosclerosis and stroke. The bacterial homologue of the human enzyme, MetF, has been found to be a useful model in genetic and biochemical studies. The similarity of Escherichia coli MetF and human MTHFR proteins is so high that particular mutations in the corresponding human gene can be reflected by the bacterial mutants. For example, the A222 V substitution in MTHFR (caused by the C667T substitution in the MTHFR gene) can be ascribed to the A117 V substitution in MetF. Here, it is reported that a temperature-sensitive MetF117 (A117 V) protein can be partially protected from a thermal inactivation by the heat shock proteins from the Hsp70/100 systems. Moreover, activity of the thermally denatured enzyme can be partially restored by the same heat shock proteins. High temperature protein G (HtpG) had no effect on MetF117 activity in both experimental systems. The presented results indicate that functions of heat shock proteins may be required for maintenance of the MetF117 function. This may have implications for the mechanisms of arteriosclerosis and stroke, especially in the light of previous findings that the A222 V MTHFR polymorphism may be a risk factor for stroke, as well as recently published results which demonstrated the increased levels of antibodies against heat shock proteins in stroke patients.

Genistein inhibits activities of methylenetetrahydrofolate reductase and lactate dehydrogenase, enzymes which use NADH as a substrate.

Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a natural isoflavone revealing many biological activities. Thus, it is considered as a therapeutic compound in as various disorders as cancer, infections and genetic diseases. Here, we demonstrate for the first time that genistein inhibits activities of bacterial methylenetetrahydrofolate reductase (MetF) and lactate dehydrogenase (LDH). Both enzymes use NADH as a substrate, and results of biochemical as well as molecular modeling studies with MetF suggest that genistein may interfere with binding of this dinucleotide to the enzyme. These results have implications for our understanding of biological functions of genistein and its effects on cellular metabolism.

Identification of QTLs affecting scopolin and scopoletin biosynthesis in Arabidopsis thaliana.

BACKGROUND: Scopoletin and its glucoside scopolin are important secondary metabolites synthesized in plants as a defense mechanism against various environmental stresses. They belong to coumarins, a class of phytochemicals with significant biological activities that is widely used in medical application and cosmetics industry. Although numerous studies showed that a variety of coumarins occurs naturally in several plant species, the details of coumarins biosynthesis and its regulation is not well understood. It was shown previously that coumarins (predominantly scopolin and scopoletin) occur in Arabidopsis thaliana (Arabidopsis) roots, but until now nothing is known about natural variation of their accumulation in this model plant. Therefore, the genetic architecture of coumarins biosynthesis in Arabidopsis has not been studied before. RESULTS: Here, the variation in scopolin and scopoletin content was assessed by comparing seven Arabidopsis accessions. Subsequently, a quantitative trait locus (QTL) mapping was performed with an Advanced Intercross Recombinant Inbred Lines (AI-RILs) mapping population EstC (Est-1 × Col). In order to reveal the genetic basis of both scopolin and scopoletin biosynthesis, two sets of methanol extracts were made from Arabidopsis roots and one set was additionally subjected to enzymatic hydrolysis prior to quantification done by high-performance liquid chromatography (HPLC). We identified one QTL for scopolin and five QTLs for scopoletin accumulation. The identified QTLs explained 13.86% and 37.60% of the observed phenotypic variation in scopolin and scopoletin content, respectively. In silico analysis of genes located in the associated QTL intervals identified a number of possible candidate genes involved in coumarins biosynthesis. CONCLUSIONS: Together, our results demonstrate for the first time that Arabidopsis is an excellent model for studying the genetic and molecular basis of natural variation in coumarins biosynthesis in plants. It additionally provides a basis for fine mapping and cloning of the genes involved in scopolin and scopoletin biosynthesis. Importantly, we have identified new loci for this biosynthetic process.

Optimization of Polydimethylsiloxane-Modified Composite Synthesis and Its Impact on Collagen Interactions: Perspectives for Biomedical Applications.

This research explores how silica composites modified with polydimethylsiloxane interact with collagen, aiming to enhance their application in the biomedical field. By adjusting the amount of polydimethylsiloxane in these composites, we evaluated their capacity to bind with collagen, an essential feature for biomaterials used in tissue engineering and drug delivery. Our findings reveal that incorporating polydimethylsiloxane into silica composites significantly boosts collagen attachment, indicating strong binding interactions. Notably, the collagen adhered to the composites maintains its natural structure, ensuring its functionality and compatibility with living tissues. This aspect is critical for biomaterials that support cell growth and regeneration in tissue scaffolds. Additionally, this study investigates how the viscosity of polydimethylsiloxane influences collagen binding, offering insights into the tailoring of composite properties for better biological performance. This work highlights the potential of polydimethylsiloxane-modified silica composites in creating innovative biomaterials for regenerative medicine and targeted therapeutic delivery.

Nigella sativa seed extract applicability in preparations against methicillin-resistant Staphylococcus aureus and effects on human dermal fibroblasts viability.

ETHNOPHARMACOLOGICAL RELEVANCE: Nigella sativa L. seed extracts and oils have been embraced by traditional medicine of cultures inhabiting Middle East and North Africa for centuries. Among other uses, it has been applied against dermatitis and eczema often worsened by staphylococcal colonization of the skin. AIM OF THE STUDY: The study was conducted to evaluate applicability of N. sativa seed extract in antibacterial skin formulations by examination of its activity against methicillin-resistant Staphylococcus aureus as well as cytotoxicity against human dermal fibroblasts. MATERIALS AND METHODS: Two variants of N. sativa seed extract containing 9.91 and 2.10 % of thymoquinone were prepared by supercritical carbon dioxide extraction. The extracts and standards of their major volatile ingredients; thymoquinone, thymol, p-cymene alongside with the reference antiseptics; chlorquinaldol and a combination of amylmetacresol with 2,4-dichlorobenzyl alcohol were subjected to evaluation of antibacterial efficacy against a collection of Staphylococcus aureus strains. The preparation based on Vaseline containing 1% of N. sativa extract was applied on Mueller-Hinton agar plates and its ability to inhibit S. aureus growth was examined. The MTT assay was employed to study cytotoxic effects of the thymoquinone-rich N. sativa seed extract against HDFa fibroblasts. RESULTS: N. sativa seed extract and thymoquinone have shown potent bacteriostatic and bactericidal effect against Staphylococcus aureus, including methicillin-resistant strains (MRSA) isolated in Poland. Results suggest that N. sativa seed extract activity against S. aureus should mainly be attributed to thymoquinone, which was effective in concentrations of 4-16 µg/ml. Regarding the activity against S. aureus, thymoquinone was more efficient than a combination of amylmetacresol with 2,4-dichlorobenzyl alcohol and comparable to chlorquinaldol. The Vaseline-based preparation containing N. sativa extract caused growth inhibition comparable to an equally concentrated DMSO solution of the extract. The IC50 of N. sativa extract against HDFa fibroblast was determined at 0.2 mg/ml, which was 2-fold higher than the average MIC and MBC of the extract against S. aureus. CONCLUSIONS: The observed effectiveness of N. sativa seed extracts against bacteria was found to be dominantly dependent on concentration of thymoquinone. Its efficiency against S. aureus isolates as well as results of cytotoxicity examination against human dermal fibroblasts indicate on its applicability as an antibacterial agent for topical use and motivates further research in this area.

Improved HPLC method for total plasma homocysteine detection and quantification.

Recent clinical research has pointed at hyperhomocysteinemia as an independent risk factor in a number of cardiovascular and neurological diseases. We have improved a chromatographic method of total plasma homocysteine measurements in order to obtain higher sensitivity, reliability and reproducibility. The method demonstrates excellent linearity (R=0.999), range (<2-100 microM), precision (instrumental RSD 0.06 and method RSD 1.17), accuracy (recovery of 99.92 and RSD 1.27), reproducibility, quantification limit and ruggedness (e.g. pH from 2.0 to 2.5). Because even a small increase in homocysteine level can be a significant risk factor of cardiovascular diseases, such a precise method is required. The constructed method allows the measurement of plasma pyridoxal phosphate, PLP, the co-enzyme form of vitamin B(6), on the same column and similar reagents. The developed method has been successfully applied to measure both total plasma and serum homocysteine in a group of acute stroke patients.

Growth of bacterial phytopathogens in animal manures.

Animal manures are routinely applied to agricultural lands to improve crop yield, but the possibility to spread bacterial phytopathogens through field fertilization has not been considered yet. We monitored 49 cattle, horse, swine, sheep or chicken manure samples collected in 14 Polish voivodeships for the most important plant pathogenic bacteria - Ralstonia solanacearum (Rsol), Xanthomonas campestris pv. campestris (Xcc), Pectobacterium carotovorum subsp. carotovorum (Pcc), Pectobacterium atrosepticum (Pba), Erwinia amylovora (Eam), Clavibacter michiganensis subsp. sepedonicus (Cms) and Dickeya sp. (Dsp). All of the tested animal fertilizers were free of these pathogens. Subsequently, the growth dynamics of Pba, Pcc, Rsol, and Xcc in cattle, horse, swine, sheep and chicken manures sterilized either by autoclaving or filtration was evaluated. The investigated phytopathogens did not exhibit any growth in the poultry manure. However, the manure filtrates originating from other animals were suitable for microbial growth, which resulted in the optical density change of 0.03-0.22 reached within 26 h (48 h Rsol, 120 h Xcc), depending on bacterial species and the manure source. Pcc and Pba multiplied most efficiently in the cattle manure filtrate. These bacteria grew faster than Rsol and Xcc in all the tested manure samples, both the filtrates and the autoclaved semi-solid ones. Though the growth dynamics of investigated strains in different animal fertilizers was unequal, all of the tested bacterial plant pathogens were proven to use cattle, horse, swine and sheep manures as the sources of nutrients. These findings may contribute to further research on the alternative routes of spread of bacterial phytopathogens, especially because of the fact that the control of pectionolytic bacteria is only based on preventive methods.

Effect of silicone on the collagen fibrillogenesis and stability.

Collagen, the most abundant protein in mammals, is able to form fibrils, which have central role in tissue repair, fibrosis, and tumor invasion. As a component of skin, tendons, and cartilages, this protein contacts with any implanted materials. An inherent problem associated with implanted prostheses is their propensity to be coated with host proteins shortly after implantation. Also, silicone implants undergoing relatively long periods of contact with blood can lead to formation of thrombi and emboli. In this paper, we demonstrate the existence of interactions between siloxanes and collagen. Low-molecular-weight cyclic siloxane (hexamethylcyclotrisiloxane-D3) and polydimethylsiloxanes (PDMS) forming linear chains, ranging in viscosity from 20 to 12,000 cSt, were analyzed. We show that D3 as well as short-chain PDMS interact with collagen, resulting in a decrease in fibrillogenesis. However, loss of collagen native structure does not occur because of these interactions. Rather, collagen seems to be sequestered in its native form in an interlayer formed by collagen-siloxane complexes. On the other hand, silicone molecules with longer chains (i.e., PDMS with viscosity of 1000 and 12,000 cSt, the highest viscosity analyzed here) demonstrate little interaction with this protein and do not seem to affect collagen activity.

Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli.

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.

Increased levels of antibodies against heat shock proteins in stroke patients.

Ischemic stroke is the second leading cause of death worldwide. One of the main risk factors of the ischemic stroke is atherosclerosis which is a chronic inflammatory and immune-mediated disease. Bacterial infections generate specific human antibodies against various antigens, including Hsps. It has been demonstrated that Hsps are selectively overexpressed in the atherosclerotic lesions. The amino acid sequence homology between human and bacterial Hsps may lead to an autoimmune response by immunological cross-reaction. Such immune response against Hsps overexpressed in the blood vessels under stressful conditions may contribute to inflammatory processes and subsequent development of atherosclerosis. In this study we determined the antibody levels against bacterial and human Hsp by ELISA in blood plasma obtained from stroke patients. Using ANOVA we analyzed levels of Hsp-antibodies in control and patient groups and correlate them with several stroke risk factors. The group of stroke patients had elevated levels of anti-Hsp antibodies compared to the control group. We also discovered an antibody level increase in patients that previously underwent another stroke. Our data provide evidence that autoimmunity could underlie formation of atherosclerosis plaque leading to stroke.

Homocysteine, heat shock proteins, genistein and vitamins in ischemic stroke--pathogenic and therapeutic implications.

Stroke is one of the most devastating neurological conditions, with an approximate worldwide mortality of 5.5 million annually and loss of 44 million disability-adjusted life-years. The etiology of stroke is often unknown; it has been estimated that the etiology and pathophysiology remains unexplained in more than 40% of stroke cases. The conventional stroke risk factors, including hypertension, diabetes mellitus, smoking, and cardiac diseases, do not fully account for the risk of stroke, and stroke victims, especially young subjects, often do not have any of these factors. It is very likely that inflammation, specific genetic predispositions and traditional risk factors interact with each other and may together increase the risk of stroke. Inflammatory and immune responses play important roles in the course of ischemic stroke. Hyperhomocysteinemia (hcy) is considered a modifiable risk factor for stroke, possibly through an atherogenic and prothrombotic mechanism. Both genetic and environmental factors (e.g., dietary intake of folic acid and B vitamins) affect homocysteine level. Identification of the role of hcy as a modifiable risk factor for stroke and of HSPs as regulators of the immune response may lead to more effective prevention and treatment of stroke through dietary and pharmacological intervention. Dietary modification may also include supplementation with novel preventive compounds, such as the antioxidative isoflavones--genistein or daidzein.

Targeting of p53 and its homolog p73 by protoporphyrin IX.

The p53 tumor suppressor is recognized as a promising target for anti-cancer therapies. We previously reported that protoporphyrin IX (PpIX) disrupts the p53/murine double minute 2 (MDM2) complex and leads to p53 accumulation and activation of apoptosis in HCT 116 cells. Here we show the direct binding of PpIX to the N-terminal domain of p53. Furthermore, we addressed the induction of apoptosis in HCT 116 p53-null cells by PpIX and revealed interactions between PpIX and p73. We propose that PpIX disrupts the p53/MDM2 or MDMX and p73/MDM2 complexes and thereby activates the p53- or p73-dependent cancer cell death.

Homocysteine level and metabolism in ischemic stroke in the population of Northern Poland.

OBJECTIVES: In this study we analyzed the occurrence of ischemic brain stroke in Northern Poland in regard to risk factors. DESIGN AND METHODS: 131 ischemic stroke patients and 64 controls were studied. Analyzed risk factors included conventional risk factors, total plasma homocysteine level and polymorphisms of the main enzymes of homocysteine metabolism-methylenetetrahydrofolate reductase (polymorphisms C677T and A1298C) and cystathionine beta synthase (polymorphism T833C). RESULTS: We confirmed the occurrence of a number of conventional risk factors in ischemic stroke. We found that hyperhomocysteinemia is an independent risk factor (p=0.0001). Plasma homocysteine correlated inversely with plasma vitamin B(6). We also found a relationship between C677T polymorphism type and hyperhomocysteinemia (p=0.0266). CONCLUSIONS: The occurrence of studied polymorphisms in the population of northern Poland was higher than reported previously for similar populations. However, none of the studied genetic factors were found to be significant risk factors in ischemic brain stroke.