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.

Abnormal Sphingolipid World in Inflammation Specific for Lysosomal Storage Diseases and Skin Disorders.

Research in recent years has shown that sphingolipids are essential signalling molecules for the proper biological and structural functioning of cells. Long-term studies on the metabolism of sphingolipids have provided evidence for their role in the pathogenesis of a number of diseases. As many inflammatory diseases, such as lysosomal storage disorders and some dermatologic diseases, including psoriasis, atopic dermatitis and ichthyoses, are associated with the altered composition and metabolism of sphingolipids, more studies precisely determining the responsibilities of these compounds for disease states are required to develop novel pharmacological treatment opportunities. It is worth emphasizing that knowledge from the study of inflammatory metabolic diseases and especially the possibility of their treatment may lead to insight into related metabolic pathways, including those involved in the formation of the epidermal barrier and providing new approaches towards workable therapies.

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.

The LA loop as an important regulatory element of the HtrA (DegP) protease from Escherichia coli: structural and functional studies.

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA' (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.

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.

Activities of genes controlling sphingolipid metabolism in human fibroblasts treated with flavonoids.

Natural flavonoids such as genistein, kaempferol and daidzein were previously found to be able to reduce efficiency of glycosaminoglycan synthesis in cells of patients suffering from mucopolysaccharidoses, inherited metabolic diseases with often brain disease symptoms. This feature was employed to test these compounds as potential drugs for treatment other neuronopathic lysosomal storage disorders, in which errors in sphingolipid metabolism occur. In this report, on the basis of DNA microarray analyses and quantitative real time PCR experiments, we present evidence that these compounds modify expression of genes coding for enzymes required for metabolism of sphingolipids in human dermal fibroblasts (HDFa). Expression of several genes involved in sphingolipid synthesis was impaired by tested flavonoids. Therefore, it is tempting to speculate that they may be considered as potential drugs in treatment of LSD, in which accumulation of sphingolipids, especially glycosphingolipids, occurs. Nevertheless, further studies on more advances models are required to test this hypothesis and to assess a therapeutic potential for flavonoids in this group of metabolic brain diseases.

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.

Factors and processes modulating phenotypes in neuronopathic lysosomal storage diseases.

Lysosomal storage diseases are inherited metabolic disorders caused by genetic defects causing deficiency of various lysosomal proteins, and resultant accumulation of non-degraded compounds. They are multisystemic diseases, and in most of them (>70%) severe brain dysfunctions are evident. However, expression of various phenotypes in particular diseases is extremely variable, from non-neuronopathic to severely neurodegenerative in the deficiency of the same enzyme. Although all lysosomal storage diseases are monogenic, clear genotype-phenotype correlations occur only in some cases. In this article, we present an overview on various factors and processes, both general and specific for certain disorders, that can significantly modulate expression of phenotypes in these diseases. On the basis of recent reports describing studies on both animal models and clinical data, we propose a hypothesis that efficiency of production of compounds that cannot be degraded due to enzyme deficiency might be especially important in modulation of phenotypes of patients suffering from lysosomal storage diseases.

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.

Non-steroidal anti-inflammatory drugs are safe with respect to the transcriptome of human dermal fibroblasts.

Non-steroidal anti-inflammatory drugs (NSAIDs) provide important benefits to millions of patients, but are associated with a number of serious adverse events. These adverse drug reactions are an important clinical issue and a serious public health risk. While most unfortunate responses in human to NSAIDs are mild and may disappear after decreasing the dose or withdrawal of the drug, some of them can produce serious outcomes. Currently, little is known regarding the effects of NSAIDs on global RNA expression in normal, non-transformed cells. Therefore, in this report, the effect of NSAIDs, COX-nonspecific and COX-2-specific inhibitors, indomethacin and nimesulide respectively, commonly used medications worldwide for the reduction of pain, fever, inflammation and stiffness, on transcriptomic signature of human dermal fibroblasts was investigated. A total of 3803 differentially expressed genes with a fold change greater than or equal to 1.3 and below than or equal to 0.7 for whole genome transcripts, with a P value of < 0.05 were identified in response to all applied conditions. We found that although the total number of deregulated genes was relatively high at such criteria, changes in fibroblast transcriptome profile after treatment at selected experimental conditions were however smallish, as the selected drugs slightly modulate transcriptome with only a few genes with expression altered a bit more than twice. Nevertheless, transcriptomic data has its own limitations and it cannot reflect all post-transcriptional changes, which in turn may cause same risks, especially for a long time of medication.

Molecular action of isoflavone genistein in the human epithelial cell line HaCaT.

Due to its strong proliferation-reducing effects on keratinocytes, and also anti-inflammatory properties, the isoflavone genistein has already been proposed as a possible antipsoriatic compound. As there is still no detailed information on this topic, we examined the effects of genistein by using an in vitro model of both, normal and "psoriasis-like" keratinocytes at this stage of our work exhaustively testing the selected flavonoid in a mono-treated experimental design. Gene expression studies revealed transcriptional changes that confirms known disease-associated pathways and highlights many psoriasis-related genes. Our results suggested that aberrant expression of genes contributing to the progress of psoriasis could be improved by the action of genistein. Genistein prevented "cytokine mix" as well as TNF-α-induced NF-κB nuclear translocation, with no effect on the PI3K signaling cascade, indicating the luck of turning this pathway into NF-κB activation. It could have attenuated TNF-α and LPS-induced inflammatory responses by suppressing ROS activation. Regardless of the type of keratinocyte stimulation used, reduction of cytokine IL-8, IL-20 and CCL2 production (both at RNA and protein level) following genistein treatment was visible. Because investigations of other groups supported our commentary on potential administration of genistein as a potential weapon in the armamentarium against psoriasis, it is believed that this paper should serve to encourage researchers to conduct further studies on this subject.

Binding of MmeI restriction-modification enzyme to its specific recognition sequence is stimulated by S-adenosyl-L-methionine.

Restriction endonucleases serve as a very good model for studying specific protein-DNA interaction. MmeI is a very interesting restriction endonuclease, but although it is useful in Serial Analysis of Gene Expression, still very little is known about the mechanism of its interaction with DNA. MmeI is a unique enzyme as besides cleaving DNA it also methylates specific sequence. For endonucleolytic activity MmeI requires Mg(II) and S-adenosyl-l-methionine (AdoMet). AdoMet is a methyl donor in the methylation reaction, but its requirement for DNA cleavage remains unclear. In the present article we investigated MmeI interaction with DNA with the use of numerous methods. Our electrophoretic mobility shift assay revealed formation of two types of specific protein-DNA complexes. We speculate that faster migrating complex consists of one protein molecule and one DNA fragment whereas, slower migrating complex, which appears in the presence of AdoMet, may be a dimer or multimer form of MmeI interacting with specific DNA. Additionally, using spectrophotometric measurements we showed that in the presence of AdoMet, MmeI protein undergoes conformational changes. We think that such change in the enzyme structure, upon addition of AdoMet, may enhance its specific binding to DNA. In the absence of AdoMet MmeI binds DNA to the much lower extent.

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria.

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

The DnaK chaperones from the archaeon Methanosarcina mazei and the bacterium Escherichia coli have different substrate specificities.

Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaK(Mm)) and the DnaK from the bacterium Escherichia coli (DnaK(Ec)) were functionally similar when assayed in vitro but DnaK(Mm) failed to substitute for DnaK(Ec) in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaK(Mm) and DnaK(Ec). DnaK(Mm) showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaK(Ec). Furthermore, it was unable to negatively regulate the E. coli sigma32 transcription factor level under heat shock conditions and poorly bound purified sigma32, which is a native substrate of DnaK(Ec). These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaK(Mm) showed some structural differences in the substrate-binding domains of DnaK(Mm) and DnaK(Ec), which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaK(Mm) was found preferably in high molecular mass oligomeric forms, contrary to DnaK(Ec). Oligomers of DnaK(Mm) could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaK(Mm).