J. Klaudíny - Academia.edu (original) (raw)

Papers by J. Klaudíny

Research paper thumbnail of Methylglyoxal-induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications

Fitoterapia, 2012

Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial com... more Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial component found in Revamil honey, peptide defensin1, was not identified in manuka honey. The primary aim of the study was to evaluate the content of defensin1 in honeys of different botanical origins and to investigate a presumed effect of reactive MGO on defensin1 and a dominant protein of honey MRJP1 in manuka honey. Immunoblotting of honey samples showed that defensin1 was a regular but quantitatively variable component of honeys. One of the reasons of varying contents of defensin1 in different honeys seems to be constitutive but varying defensin1 expression in individual honeybees in bee populations that we documented on samples of nurse and forager bees by RT-PCR. Comparative analyses of honeys revealed a size modification of defensin1, MRJP1 and probably also α-glucosidase in manuka honey. We further showed that (i) the treatment of purified defensin1 in solution containing high amount of MGO caused a time-dependent loss of its antibacterial activity and (ii) increasing MGO concentrations in a non-manuka honey were connected with a gradual increase in the molecular weight of MRJP1. Obtained results demonstrate that MGO abrogates the antibacterial activity of defensin1 and modifies MRJP1 in manuka honey. We assume that MGO could also have negative effects on the structure and function of other proteins/peptides in manuka honey, including glucose oxidase, generating hydrogen peroxide.

Research paper thumbnail of The sequence of Japanese quail ovalbumin cDNA

Nucleic Acids …, 1990

The cDNA clone, encoding 383 amino acid residues was obtained from oviduct cDNA library prepared ... more The cDNA clone, encoding 383 amino acid residues was obtained from oviduct cDNA library prepared from 1.5-2.5 kb cDNA fraction (1). Comparing the amino acid (aa) residues derived from the quail cDNA with known protein sequence of hen and chicken ovalbumin (2, 3) the substitution of 42 aa and the deletion of 3aa was determined.

Research paper thumbnail of Characterization of a long-chain α-galactosidase from Papiliotrema flavescens

World Journal of Microbiology and Biotechnology

Research paper thumbnail of Antibacterial Activity of Different Blossom Honeys: New Findings

Molecules

Antibacterial activity is the most investigated biological property of honey. The goal of this st... more Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H2O2), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H2O2 and TP in honeys. A strong correlation was found between the H2O2 and TP content. On the other hand, no correlation was found between the ...

Research paper thumbnail of 10-HDA, A Major Fatty Acid of Royal Jelly, Exhibits pH Dependent Growth-Inhibitory Activity Against Different Strains of Paenibacillus larvae

Molecules

Paenibacillus larvae (P. larvae) is a bacterial pathogen causing American foulbrood (AFB), the mo... more Paenibacillus larvae (P. larvae) is a bacterial pathogen causing American foulbrood (AFB), the most serious disease of honeybee larvae. The food of young larvae could play an important role in the resistance of larvae against AFB. It contains antibacterial substances produced by honeybees that may inhibit the propagation of the pathogen in larval midguts. In this study, we identified and investigated the antibacterial effects of one of these substances, trans-10-hydroxy-2-decenoic acid (10-HDA), against P. larvae strains including all Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes. Its inhibitory activities were studied by determining the minimum inhibitory concentrations (MICs). It was found that 10-HDA efficacy increases substantially with decreasing pH; up to 12-fold differences in efficacy were observed between pH = 5.5 and pH = 7.2. P. larvae strains showed different susceptibility to 10-HDA; up to 2.97-fold differences existed among various strains with envir...

Research paper thumbnail of Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo

Scientific Reports

Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects... more Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects, including antibacterial, anti-inflammatory and immunomodulatory activities, in various cell types. However, no component(s) (other than antibacterial) have been identified in RJ-accelerated wound healing. In this study, we demonstrate that keratinocytes are responsible for the elevated production of matrix metalloproteinase-9 (MMP-9) after incubation with a water extract of RJ. Furthermore, the keratinocyte migration and wound closure rates were significantly increased in the presence of RJ extract. MMP-9 production was reduced significantly following proteinase K treatment but remained stable after heat treatment, indicating that active component(s) have a proteinous character. To identify the component responsible for inducing MMP-9 production, RJ extract was fractionated using C18 RP-HPLC. In fractions exhibiting stimulatory activity, we immunochemically detected the bee-derived antibacterial peptide, defensin-1. Defensin-1 was cloned, and recombinant peptide was produced in a baculoviral expression system. Defensin-1 stimulated MMP-9 secretion from keratinocytes and increased keratinocyte migration and wound closure in vitro. In addition, defensin-1 promoted re-epithelisation and wound closure in uninfected excision wounds. These data indisputably demonstrate that defensin-1, a regular but concentration variable factor found in honey and RJ, contributes to cutaneous wound closure by enhancing keratinocyte migration and MMP-9 secretion. Keratinocytes, a major cellular component of the epidermis, are responsible for restoring the epidermis after injury through a process termed epithelialisation. The migration, proliferation, and differentiation of fibroblasts and keratinocytes, as well as interactions between these cells are critical for effective re-epithelialisation and wound healing. Immature keratinocytes produce matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, and plasmin, which enables their dissociation from the basement membrane and facilitates their migration. MMP-9 (gelatinase B) is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of extracellular matrix proteins, such as type III and IV collagens and elastin. MMP-9 plays an important role in normal wound healing, particularly related to extracellular matrix (ECM) remodelling and re-epithelialisation. Wound healing is impaired when MMP-9 is inhibited 1, 2. Historically, honeybee products, such as honey and royal jelly (RJ), have been used to treat a broad spectrum of injuries. RJ is part of the diet of honeybee larvae and is secreted from the hypopharyngeal and mandibular glands of worker honey bees 3. RJ has been used since ancient times to facilitate wound healing. RJ acts as an antimicrobial 4-8 and antioxidative agent 9 and as an immunomodulator with anti-inflammatory properties 10, 11. The topical application of RJ to treat human diabetic foot ulcers 12-14 provides compelling evidence that RJ can accelerate wound healing. Furthermore, RJ promotes wound healing in an animal model of uninfected wound 15. However, the mechanisms of action, other than antibacterial effects, associated with the effects of RJ

Research paper thumbnail of Newly discovered features of the updated sequence of royal jelly protein RJP571; longer repetitive region on C-terminus and homology toDrosophila melanogasteryellow protein

Journal of Apicultural Research, 1996

Research paper thumbnail of Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase from Apis mellifera

Acta Crystallographica Section F Structural Biology and Crystallization Communications, Nov 1, 2012

Research paper thumbnail of The MRJP/YELLOW protein family of Apis mellifera: Identification of new members in the EST library

Journal of Insect Physiology, Jan 31, 2004

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Droso... more Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.

Research paper thumbnail of MRJP9, an ancient protein of the honeybee MRJP family with non-nutritional function

J Apicult Res, 2007

Major royal jelly proteins (MRJPs) form a subfamily within a larger Yellow/MRJP family of protein... more Major royal jelly proteins (MRJPs) form a subfamily within a larger Yellow/MRJP family of proteins. Whereas multiple Yellow-like proteins have been found in all insect genomes including honeybee, MRJPs seem to be restricted to honeybees. Eight MRJP proteins, termed MRJP1-8, have been characterized so far. We found that proteomic analyses of bee hypopharyngeal glands and venom sac identified proteolytic fragments of a protein, which might be a novel member of the MRJP family. Using this information and availability of honeybee genome, we cloned the corresponding cDNA and confirmed by sequencing that it encodes an uncharacterized MRJP9 protein. Interestingly, a cDNA coding for a relative of MRJP9 was found in paper wasp EST library. By phylogenetic analyses we show that MRJP9 is an ancient member of the MRJP family, which diverged prior to occurrence of nutritionally valuable nitrogen-rich repeat regions in MRJPs. Identification of MRJP9 in several bee organs, its comparatively low expression levels there, together with the existence of a MRJP-like protein in wasp indicate that it performs function(s) different from larval nutrition. A novel hypothesis on possible role of MRJP9 found in bee venom as a possible immunosensitizing factor contributing to allergy against royal jelly is discussed.

Research paper thumbnail of Honeybee defensins: their characterization and studies of their expression in honeybee colonies

Collection Symposium Series, 2015

Research paper thumbnail of Cloning of Japanese quail ovalbumin cDNA in E. coli

Folia biologica

Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using ... more Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease. The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA. The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed. An atypical strategy was used for cloning full-length cDNAov. The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes. A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E. coli DHl cells. Seventy-two clones were screened for the presence of full-length cDNAov, ...

Research paper thumbnail of Complete sequence of the coding region of human elongation factor 2 (EF-2) by enzymatic amplification of cDNA from human ovarian granulosa cells

Biological chemistry Hoppe-Seyler, 1989

The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starti... more The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starting with total double-stranded cDNA from human ovarian granulosa cells. The amplified DNA fragment with a length of 1765 bp was restricted and sequenced by the shot gun approach. From the sequences obtained from the amplified fragment and the cDNA insert of pHGR81 [Rapp et al. (1988) Biol. Chem. Hoppe-Seyler 369, 247-250] respectively, the DNA sequence containing the complete coding as well as the 3'-untranslated region was assembled.

Research paper thumbnail of Honeybee glucose oxidase—its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys

Naturwissenschaften, 2014

Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2),... more Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2), which is generated by glucose oxidase (GOX)-mediated conversion of glucose in diluted honey. However, honeys exhibit considerable variation in their antibacterial activity. Therefore, the aim of the study was to identify the mechanism behind the variation in this activity and in the H2O2 content in honeys associated with the role of GOX in this process. Immunoblots and in situ hybridization analyses demonstrated that gox is solely expressed in the hypopharyngeal glands of worker bees performing various tasks and not in other glands or tissues. Real-time PCR with reference genes selected for worker heads shows that the gox expression progressively increases with ageing of the youngest bees and nurses and reached the highest values in processor bees. Immunoblot analysis of honey samples revealed that GOX is a regular honey component but its content significantly varied among honeys. Neither botanical source nor geographical origin of honeys affected the level of GOX suggesting that some other factors such as honeybee nutrition and/or genetic/epigenetic factors may take part in the observed variation. A strong correlation was found between the content of GOX and the level of generated H2O2 in honeys except honeydew honeys. Total antibacterial activity of most honey samples against Pseudomonas aeruginosa isolate significantly correlated with the H2O2 content. These results demonstrate that the level of GOX can significantly affect the total antibacterial activity of honey. They also support an idea that breeding of novel honeybee lines expressing higher amounts of GOX could help to increase the antibacterial efficacy of the hypopharyngeal gland secretion that could have positive influence on a resistance of colonies against bacterial pathogens.

Research paper thumbnail of A simple, non-radioactive DNA fingerprinting method for identifying patrilines in honeybee colonies

Apidologie, 1998

Prinrcrs were derived flanking a rniclosatcllite motil'of the cloned Z-locus. The PCR product of ... more Prinrcrs were derived flanking a rniclosatcllite motil'of the cloned Z-locus. The PCR product of the Z-locLrs was variablc in size and up to four alleles were fbund in a sarnple ol l I wurkers within onc colony. Using the corlbination of thlee loci. the Z. the Q (both linkecl to thc sex locus) and a rcyal .jcllv protein gene (RJP57-l) we were able to discrinrinate fivc patlilines in the 1l worker sample. Using the '"vcll cstablishcd rniclosatcllite tcchuolo-rr1. howcvet. seven and six patrilincs could be identitied. The technique may cnablc labolatories which iack an isotope facilitl,and ccluipped with onlv a PCIR thcr-rnocvclel and agarose gel apparatus to studv thc polvanclrous mating s1'stcm ofthc honer'tree in rr rarietv o1'diflerent contexts. O Inra/DIB/AGlIl/Elsevier. Paris fingerprinting / patriline / rnating / honel'bee I Apis melliJbra I l?CR

Research paper thumbnail of Anti-biofilm Effects of Honey Against Wound Pathogens Proteus mirabilis and Enterobacter cloacae

Phytotherapy Research, 2014

Biofilm growth and its persistence within wounds have recently been suggested as contributing fac... more Biofilm growth and its persistence within wounds have recently been suggested as contributing factors to impaired healing. The goal of this study was to investigate the anti-biofilm effects of several honey samples of different botanical origin, including manuka honey against Proteus mirabilis and Enterobacter cloacae wound isolates. Quantification of biofilm formation was carried out using a microtiter plate assay. All honeys at a sub-inhibitory concentration of 10% (w/v) significantly reduced the biofilm development of both isolates. Similarly, at a concentration of 50% (w/v), each of the honeys caused significant partial detachment of Pr. mirabilis biofilm after 24 h. On the other hand, no honey was able to significantly detach Ent. cloacae biofilm. In addition, treatment of Ent. cloacae and Pr. mirabilis biofilms with all honeys resulted in a significant decrease in colony-forming units per well values in a range of 0.35-1.16 and 1.2-7.5 log units, respectively. Of the tested honeys, manuka honey possessed the most potent anti-biofilm properties. Furthermore, methylglyoxal, an antibacterial compound of manuka honey, was shown to be responsible for killing biofilm-embedded wound bacteria. These findings suggest that manuka honey could be used as a potential therapy for the treatment of wounds containing Pr. mirabilis or Ent. cloacae.

Research paper thumbnail of Expression of a part of the Potato virus A non-structural protein P3 in Escherichia coli for the purpose of antibody preparation and P3 immunodetection in plant material

Journal of Virological Methods, 2006

The N-terminal part of the Potato virus A (PVA) P3 protein was cloned into two E. coli fusion exp... more The N-terminal part of the Potato virus A (PVA) P3 protein was cloned into two E. coli fusion expression systems. An overexpression of the P3 fragment fused with thioredoxin was observed between 2 and 21 h after induction. The protein formed insoluble inclusions. Decreasing the cultivation temperature did not enhance its solubility. To obtain antigen for antibody preparation, inclusions were concentrated and purified by sucrose gradient centrifugation, and subjected to SDS-polyacrylamide gel electrophoresis. The band specific for the protein was excised from the gel and used for rabbit immunization. Obtained antibody tested positive with high specificity in immunoblots of expressed PVA P3 fused with either thioredoxin or GST. The antibody was also applied for the detection of P3 protein in plant material by immunoblot. Previous plant sap concentration was essential for most samples. Three concentration methods were tested: simple centrifugal size-exclusion filtration, the same preceded with highspeed centrifugation at 250,000 × g, and differential ammonium sulfate precipitation. The last approach was the most convenient. Plants tested included PVA P3-transgenic tobacco lines as well as PVA-infected wild-type tobacco. In all cases, mature P3 with a molecular mass of 40 kDa was detected.

Research paper thumbnail of Characterization by cDNA Cloning of Two New Human Protein Kinases

Journal of Molecular Biology, 1994

Research paper thumbnail of Methylglyoxal May Affect Hydrogen Peroxide Accumulation in Manuka Honey Through the Inhibition of Glucose Oxidase

Journal of Medicinal Food, 2014

Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it do... more Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H2O2 accumulation in natural non-manuka honeys. H2O2 concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H2O2 producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H2O2 concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8-9.1 lM H2O2 in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9-6.8 and 495.8-9.1 lM H2O2, respectively. Levels of H2O2 increased significantly over time in both honey solutions. Contrary to this, the MGOtreated honeys generated significantly lower amounts of H2O2 (P < .001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H2O2 generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.

Research paper thumbnail of The MRJP/YELLOW protein family of Apis mellifera: Identification of new members in the EST library

Journal of Insect Physiology, 2004

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Droso... more Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.

Research paper thumbnail of Methylglyoxal-induced modifications of significant honeybee proteinous components in manuka honey: Possible therapeutic implications

Fitoterapia, 2012

Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial com... more Methylglyoxal (MGO) is a major antibacterial component of manuka honey. Another antibacterial component found in Revamil honey, peptide defensin1, was not identified in manuka honey. The primary aim of the study was to evaluate the content of defensin1 in honeys of different botanical origins and to investigate a presumed effect of reactive MGO on defensin1 and a dominant protein of honey MRJP1 in manuka honey. Immunoblotting of honey samples showed that defensin1 was a regular but quantitatively variable component of honeys. One of the reasons of varying contents of defensin1 in different honeys seems to be constitutive but varying defensin1 expression in individual honeybees in bee populations that we documented on samples of nurse and forager bees by RT-PCR. Comparative analyses of honeys revealed a size modification of defensin1, MRJP1 and probably also α-glucosidase in manuka honey. We further showed that (i) the treatment of purified defensin1 in solution containing high amount of MGO caused a time-dependent loss of its antibacterial activity and (ii) increasing MGO concentrations in a non-manuka honey were connected with a gradual increase in the molecular weight of MRJP1. Obtained results demonstrate that MGO abrogates the antibacterial activity of defensin1 and modifies MRJP1 in manuka honey. We assume that MGO could also have negative effects on the structure and function of other proteins/peptides in manuka honey, including glucose oxidase, generating hydrogen peroxide.

Research paper thumbnail of The sequence of Japanese quail ovalbumin cDNA

Nucleic Acids …, 1990

The cDNA clone, encoding 383 amino acid residues was obtained from oviduct cDNA library prepared ... more The cDNA clone, encoding 383 amino acid residues was obtained from oviduct cDNA library prepared from 1.5-2.5 kb cDNA fraction (1). Comparing the amino acid (aa) residues derived from the quail cDNA with known protein sequence of hen and chicken ovalbumin (2, 3) the substitution of 42 aa and the deletion of 3aa was determined.

Research paper thumbnail of Characterization of a long-chain α-galactosidase from Papiliotrema flavescens

World Journal of Microbiology and Biotechnology

Research paper thumbnail of Antibacterial Activity of Different Blossom Honeys: New Findings

Molecules

Antibacterial activity is the most investigated biological property of honey. The goal of this st... more Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H2O2), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H2O2 and TP in honeys. A strong correlation was found between the H2O2 and TP content. On the other hand, no correlation was found between the ...

Research paper thumbnail of 10-HDA, A Major Fatty Acid of Royal Jelly, Exhibits pH Dependent Growth-Inhibitory Activity Against Different Strains of Paenibacillus larvae

Molecules

Paenibacillus larvae (P. larvae) is a bacterial pathogen causing American foulbrood (AFB), the mo... more Paenibacillus larvae (P. larvae) is a bacterial pathogen causing American foulbrood (AFB), the most serious disease of honeybee larvae. The food of young larvae could play an important role in the resistance of larvae against AFB. It contains antibacterial substances produced by honeybees that may inhibit the propagation of the pathogen in larval midguts. In this study, we identified and investigated the antibacterial effects of one of these substances, trans-10-hydroxy-2-decenoic acid (10-HDA), against P. larvae strains including all Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes. Its inhibitory activities were studied by determining the minimum inhibitory concentrations (MICs). It was found that 10-HDA efficacy increases substantially with decreasing pH; up to 12-fold differences in efficacy were observed between pH = 5.5 and pH = 7.2. P. larvae strains showed different susceptibility to 10-HDA; up to 2.97-fold differences existed among various strains with envir...

Research paper thumbnail of Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo

Scientific Reports

Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects... more Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects, including antibacterial, anti-inflammatory and immunomodulatory activities, in various cell types. However, no component(s) (other than antibacterial) have been identified in RJ-accelerated wound healing. In this study, we demonstrate that keratinocytes are responsible for the elevated production of matrix metalloproteinase-9 (MMP-9) after incubation with a water extract of RJ. Furthermore, the keratinocyte migration and wound closure rates were significantly increased in the presence of RJ extract. MMP-9 production was reduced significantly following proteinase K treatment but remained stable after heat treatment, indicating that active component(s) have a proteinous character. To identify the component responsible for inducing MMP-9 production, RJ extract was fractionated using C18 RP-HPLC. In fractions exhibiting stimulatory activity, we immunochemically detected the bee-derived antibacterial peptide, defensin-1. Defensin-1 was cloned, and recombinant peptide was produced in a baculoviral expression system. Defensin-1 stimulated MMP-9 secretion from keratinocytes and increased keratinocyte migration and wound closure in vitro. In addition, defensin-1 promoted re-epithelisation and wound closure in uninfected excision wounds. These data indisputably demonstrate that defensin-1, a regular but concentration variable factor found in honey and RJ, contributes to cutaneous wound closure by enhancing keratinocyte migration and MMP-9 secretion. Keratinocytes, a major cellular component of the epidermis, are responsible for restoring the epidermis after injury through a process termed epithelialisation. The migration, proliferation, and differentiation of fibroblasts and keratinocytes, as well as interactions between these cells are critical for effective re-epithelialisation and wound healing. Immature keratinocytes produce matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, and plasmin, which enables their dissociation from the basement membrane and facilitates their migration. MMP-9 (gelatinase B) is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of extracellular matrix proteins, such as type III and IV collagens and elastin. MMP-9 plays an important role in normal wound healing, particularly related to extracellular matrix (ECM) remodelling and re-epithelialisation. Wound healing is impaired when MMP-9 is inhibited 1, 2. Historically, honeybee products, such as honey and royal jelly (RJ), have been used to treat a broad spectrum of injuries. RJ is part of the diet of honeybee larvae and is secreted from the hypopharyngeal and mandibular glands of worker honey bees 3. RJ has been used since ancient times to facilitate wound healing. RJ acts as an antimicrobial 4-8 and antioxidative agent 9 and as an immunomodulator with anti-inflammatory properties 10, 11. The topical application of RJ to treat human diabetic foot ulcers 12-14 provides compelling evidence that RJ can accelerate wound healing. Furthermore, RJ promotes wound healing in an animal model of uninfected wound 15. However, the mechanisms of action, other than antibacterial effects, associated with the effects of RJ

Research paper thumbnail of Newly discovered features of the updated sequence of royal jelly protein RJP571; longer repetitive region on C-terminus and homology toDrosophila melanogasteryellow protein

Journal of Apicultural Research, 1996

Research paper thumbnail of Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine β-synthase from Apis mellifera

Acta Crystallographica Section F Structural Biology and Crystallization Communications, Nov 1, 2012

Research paper thumbnail of The MRJP/YELLOW protein family of Apis mellifera: Identification of new members in the EST library

Journal of Insect Physiology, Jan 31, 2004

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Droso... more Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.

Research paper thumbnail of MRJP9, an ancient protein of the honeybee MRJP family with non-nutritional function

J Apicult Res, 2007

Major royal jelly proteins (MRJPs) form a subfamily within a larger Yellow/MRJP family of protein... more Major royal jelly proteins (MRJPs) form a subfamily within a larger Yellow/MRJP family of proteins. Whereas multiple Yellow-like proteins have been found in all insect genomes including honeybee, MRJPs seem to be restricted to honeybees. Eight MRJP proteins, termed MRJP1-8, have been characterized so far. We found that proteomic analyses of bee hypopharyngeal glands and venom sac identified proteolytic fragments of a protein, which might be a novel member of the MRJP family. Using this information and availability of honeybee genome, we cloned the corresponding cDNA and confirmed by sequencing that it encodes an uncharacterized MRJP9 protein. Interestingly, a cDNA coding for a relative of MRJP9 was found in paper wasp EST library. By phylogenetic analyses we show that MRJP9 is an ancient member of the MRJP family, which diverged prior to occurrence of nutritionally valuable nitrogen-rich repeat regions in MRJPs. Identification of MRJP9 in several bee organs, its comparatively low expression levels there, together with the existence of a MRJP-like protein in wasp indicate that it performs function(s) different from larval nutrition. A novel hypothesis on possible role of MRJP9 found in bee venom as a possible immunosensitizing factor contributing to allergy against royal jelly is discussed.

Research paper thumbnail of Honeybee defensins: their characterization and studies of their expression in honeybee colonies

Collection Symposium Series, 2015

Research paper thumbnail of Cloning of Japanese quail ovalbumin cDNA in E. coli

Folia biologica

Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using ... more Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease. The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA. The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed. An atypical strategy was used for cloning full-length cDNAov. The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes. A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E. coli DHl cells. Seventy-two clones were screened for the presence of full-length cDNAov, ...

Research paper thumbnail of Complete sequence of the coding region of human elongation factor 2 (EF-2) by enzymatic amplification of cDNA from human ovarian granulosa cells

Biological chemistry Hoppe-Seyler, 1989

The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starti... more The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starting with total double-stranded cDNA from human ovarian granulosa cells. The amplified DNA fragment with a length of 1765 bp was restricted and sequenced by the shot gun approach. From the sequences obtained from the amplified fragment and the cDNA insert of pHGR81 [Rapp et al. (1988) Biol. Chem. Hoppe-Seyler 369, 247-250] respectively, the DNA sequence containing the complete coding as well as the 3'-untranslated region was assembled.

Research paper thumbnail of Honeybee glucose oxidase—its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys

Naturwissenschaften, 2014

Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2),... more Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2), which is generated by glucose oxidase (GOX)-mediated conversion of glucose in diluted honey. However, honeys exhibit considerable variation in their antibacterial activity. Therefore, the aim of the study was to identify the mechanism behind the variation in this activity and in the H2O2 content in honeys associated with the role of GOX in this process. Immunoblots and in situ hybridization analyses demonstrated that gox is solely expressed in the hypopharyngeal glands of worker bees performing various tasks and not in other glands or tissues. Real-time PCR with reference genes selected for worker heads shows that the gox expression progressively increases with ageing of the youngest bees and nurses and reached the highest values in processor bees. Immunoblot analysis of honey samples revealed that GOX is a regular honey component but its content significantly varied among honeys. Neither botanical source nor geographical origin of honeys affected the level of GOX suggesting that some other factors such as honeybee nutrition and/or genetic/epigenetic factors may take part in the observed variation. A strong correlation was found between the content of GOX and the level of generated H2O2 in honeys except honeydew honeys. Total antibacterial activity of most honey samples against Pseudomonas aeruginosa isolate significantly correlated with the H2O2 content. These results demonstrate that the level of GOX can significantly affect the total antibacterial activity of honey. They also support an idea that breeding of novel honeybee lines expressing higher amounts of GOX could help to increase the antibacterial efficacy of the hypopharyngeal gland secretion that could have positive influence on a resistance of colonies against bacterial pathogens.

Research paper thumbnail of A simple, non-radioactive DNA fingerprinting method for identifying patrilines in honeybee colonies

Apidologie, 1998

Prinrcrs were derived flanking a rniclosatcllite motil'of the cloned Z-locus. The PCR product of ... more Prinrcrs were derived flanking a rniclosatcllite motil'of the cloned Z-locus. The PCR product of the Z-locLrs was variablc in size and up to four alleles were fbund in a sarnple ol l I wurkers within onc colony. Using the corlbination of thlee loci. the Z. the Q (both linkecl to thc sex locus) and a rcyal .jcllv protein gene (RJP57-l) we were able to discrinrinate fivc patlilines in the 1l worker sample. Using the '"vcll cstablishcd rniclosatcllite tcchuolo-rr1. howcvet. seven and six patrilincs could be identitied. The technique may cnablc labolatories which iack an isotope facilitl,and ccluipped with onlv a PCIR thcr-rnocvclel and agarose gel apparatus to studv thc polvanclrous mating s1'stcm ofthc honer'tree in rr rarietv o1'diflerent contexts. O Inra/DIB/AGlIl/Elsevier. Paris fingerprinting / patriline / rnating / honel'bee I Apis melliJbra I l?CR

Research paper thumbnail of Anti-biofilm Effects of Honey Against Wound Pathogens Proteus mirabilis and Enterobacter cloacae

Phytotherapy Research, 2014

Biofilm growth and its persistence within wounds have recently been suggested as contributing fac... more Biofilm growth and its persistence within wounds have recently been suggested as contributing factors to impaired healing. The goal of this study was to investigate the anti-biofilm effects of several honey samples of different botanical origin, including manuka honey against Proteus mirabilis and Enterobacter cloacae wound isolates. Quantification of biofilm formation was carried out using a microtiter plate assay. All honeys at a sub-inhibitory concentration of 10% (w/v) significantly reduced the biofilm development of both isolates. Similarly, at a concentration of 50% (w/v), each of the honeys caused significant partial detachment of Pr. mirabilis biofilm after 24 h. On the other hand, no honey was able to significantly detach Ent. cloacae biofilm. In addition, treatment of Ent. cloacae and Pr. mirabilis biofilms with all honeys resulted in a significant decrease in colony-forming units per well values in a range of 0.35-1.16 and 1.2-7.5 log units, respectively. Of the tested honeys, manuka honey possessed the most potent anti-biofilm properties. Furthermore, methylglyoxal, an antibacterial compound of manuka honey, was shown to be responsible for killing biofilm-embedded wound bacteria. These findings suggest that manuka honey could be used as a potential therapy for the treatment of wounds containing Pr. mirabilis or Ent. cloacae.

Research paper thumbnail of Expression of a part of the Potato virus A non-structural protein P3 in Escherichia coli for the purpose of antibody preparation and P3 immunodetection in plant material

Journal of Virological Methods, 2006

The N-terminal part of the Potato virus A (PVA) P3 protein was cloned into two E. coli fusion exp... more The N-terminal part of the Potato virus A (PVA) P3 protein was cloned into two E. coli fusion expression systems. An overexpression of the P3 fragment fused with thioredoxin was observed between 2 and 21 h after induction. The protein formed insoluble inclusions. Decreasing the cultivation temperature did not enhance its solubility. To obtain antigen for antibody preparation, inclusions were concentrated and purified by sucrose gradient centrifugation, and subjected to SDS-polyacrylamide gel electrophoresis. The band specific for the protein was excised from the gel and used for rabbit immunization. Obtained antibody tested positive with high specificity in immunoblots of expressed PVA P3 fused with either thioredoxin or GST. The antibody was also applied for the detection of P3 protein in plant material by immunoblot. Previous plant sap concentration was essential for most samples. Three concentration methods were tested: simple centrifugal size-exclusion filtration, the same preceded with highspeed centrifugation at 250,000 × g, and differential ammonium sulfate precipitation. The last approach was the most convenient. Plants tested included PVA P3-transgenic tobacco lines as well as PVA-infected wild-type tobacco. In all cases, mature P3 with a molecular mass of 40 kDa was detected.

Research paper thumbnail of Characterization by cDNA Cloning of Two New Human Protein Kinases

Journal of Molecular Biology, 1994

Research paper thumbnail of Methylglyoxal May Affect Hydrogen Peroxide Accumulation in Manuka Honey Through the Inhibition of Glucose Oxidase

Journal of Medicinal Food, 2014

Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it do... more Although hydrogen peroxide (H2O2) is one of the major antibacterial factors in most honeys, it does not accumulate in medical-grade manuka honey. The goal of this study was to investigate the effect of artificially added methylglyoxal (MGO) on H2O2 accumulation in natural non-manuka honeys. H2O2 concentrations in the honey solutions were determined using a fluorimetric assay. Two, the most potent H2O2 producers honeydew honeys were mixed with MGO at final concentrations of 250, 500, and 1000 mg/kg, and incubated for 4 days at 37°C. Subsequently, H2O2 concentrations were determined in 50% (wt/vol) MGO supplemented honey solutions. In vitro crosslinking of the enzyme glucose oxidase (GOX) after incubation with MGO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tested honeys at a concentration of 50% (wt/vol) accumulated up to 495.8-9.1 lM H2O2 in 24 h. The most potent producers were the two honeydew honeys, whose 50% solutions accumulated 306.9-6.8 and 495.8-9.1 lM H2O2, respectively. Levels of H2O2 increased significantly over time in both honey solutions. Contrary to this, the MGOtreated honeys generated significantly lower amounts of H2O2 (P < .001), and this reduction was dose dependent. In addition, MGO-treated GOX formed high molecular weight adducts with increasing time of incubation accompanied by loss of its enzymatic activity. High levels of MGO in manuka honey, by modifying the enzyme GOX, might be responsible for suppressing H2O2 generation. These data highlight the detrimental effect of MGO on significant proteinaceous components of manuka honey.

Research paper thumbnail of The MRJP/YELLOW protein family of Apis mellifera: Identification of new members in the EST library

Journal of Insect Physiology, 2004

Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Droso... more Major royal jelly proteins (named MRJP1-5) of honeybee (Apis mellifera), yellow proteins of Drosophila, together with putative proteins found in several bacteria, form a protein family termed the MRJP/yellow family. Members of the family exert diverse physiological functions and amongst eukaryotes appear to be restricted to the order Insecta. MRJPs constitute about 90% of total protein of royal jelly, which is secreted by nurse bees to feed the queen and growing larvae. We looked for mrjp and yellow homologues in a honeybee brain expressed sequence tags (EST) library. In addition to the five mrjp cDNAs previously characterized, we found three additional cDNAs encoding novel MRJPs and importantly, two cDNAs coding for orthologues of Drosophila yellow proteins. One yellow cDNA and all three cDNAs coding for the novel MRJPs were assembled completely, the sequence of the other yellow homologue was partially assembled. The data we present here supports the view that repeated duplications and functional divergence occurred during the evolution of MRJPs in honeybees, with even closely related MRJPs appearing to perform diverse physiological functions. Conversely, yellow protein orthologues appear to be conserved and thus candidates for maintaining the former function(s) of yellow proteins.