"Tear down that wall"-a critical evaluation of bioinformatic resources available for lysin researchers.

Lytic enzymes, or lysins for short, break down peptidoglycan and interrupt the continuity of the cell wall, which, in turn, causes osmotic lysis of the bacterium. Their ability to destroy bacteria from within makes them promising antimicrobial agents that can be used as alternatives or supplements to antibiotics. In this paper, we briefly summarize basic terms and concepts used to describe lysin sequences and delineate major lysin groups. More importantly, we describe the domain repertoire found in lysins and critically review bioinformatic tools or databases which are used in studies of these enzymes (with particular emphasis on the repositories of Hidden Markov models). Finally, we present a novel comprehensive, meticulously curated set of lysin-related family and domain models, sort them into clusters that reflect major families, and demonstrate that the selected models can be used to efficiently search for new lysins.

The Greater Celandine: Identification and Characterization of an Antimicrobial Peptide from Chelidonium majus.

Chelidonium majus, known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein, C. majus aerial tissue is mined for the presence of antimicrobial peptides. A highly abundant cysteine-rich peptide with a length of 25 amino acids, deemed CM-AMP1, is characterized through multiple mass spectrometric approaches. Electron-activated dissociation is leveraged to differentiate between isoleucine and leucine residues and complement conventional collision-induced dissociation to gain full sequence coverage of the full-length peptide. CM-AMP1 shares little sequence similarity with any proteins in publicly available databases, highlighting the novelty of its cysteine landscape and core motif. The presence of three disulfide bonds in the native peptide confers proteolytic stability, and antimicrobial activity is greatly decreased upon the alkylation of the cysteine residues. Synthetic variants of CM-AMP1 are used to confirm the activity of the full-length sequence and the core motif. To assess the biological impact, E. coli was grown in a sublethal concentration of CM-AMP1 and quantitative proteomics was used to identify proteins produced by the bacteria under stress, ultimately suggesting a membrane lytic antimicrobial mechanism of action. This study integrates multiple analytical methods for molecular and biological characterization of a unique antimicrobial peptide identified from C. majus.

Comparative Analysis of the Placental Microbiome in Pregnancies with Late Fetal Growth Restriction versus Physiological Pregnancies.

A comparative analysis of the placental microbiome in pregnancies with late fetal growth restriction (FGR) was performed with normal pregnancies to assess the impact of bacteria on placental development and function. The presence of microorganisms in the placenta, amniotic fluid, fetal membranes and umbilical cord blood throughout pregnancy disproves the theory of the "sterile uterus". FGR occurs when the fetus is unable to follow a biophysically determined growth path. Bacterial infections have been linked to maternal overproduction of pro-inflammatory cytokines, as well as various short- and long-term problems. Proteomics and bioinformatics studies of placental biomass allowed the development of new diagnostic options. In this study, the microbiome of normal and FGR placentas was analyzed by LC-ESI-MS/MS mass spectrometry, and the bacteria present in both placentas were identified by analysis of a set of bacterial proteins. Thirty-six pregnant Caucasian women participated in the study, including 18 women with normal pregnancy and eutrophic fetuses (EFW > 10th percentile) and 18 women with late FGR diagnosed after 32 weeks of gestation. Based on the analysis of the proteinogram, 166 bacterial proteins were detected in the material taken from the placentas in the study group. Of these, 21 proteins had an exponentially modified protein abundance index (emPAI) value of 0 and were not included in further analysis. Of the remaining 145 proteins, 52 were also present in the material from the control group. The remaining 93 proteins were present only in the material collected from the study group. Based on the proteinogram analysis, 732 bacterial proteins were detected in the material taken from the control group. Of these, 104 proteins had an emPAI value of 0 and were not included in further analysis. Of the remaining 628 proteins, 52 were also present in the material from the study group. The remaining 576 proteins were present only in the material taken from the control group. In both groups, we considered the result of ns prot ≥ 60 as the cut-off value for the agreement of the detected protein with its theoretical counterpart. Our study found significantly higher emPAI values of proteins representative of the following bacteria: Actinopolyspora erythraea, Listeria costaricensis, E. coli, Methylobacterium, Acidobacteria bacterium, Bacteroidetes bacterium, Paenisporsarcina sp., Thiodiazotropha endol oripes and Clostridiales bacterium. On the other hand, in the control group statistically more frequently, based on proteomic data, the following were found: Flavobacterial bacterium, Aureimonas sp. and Bacillus cereus. Our study showed that placental dysbiosis may be an important factor in the etiology of FGR. The presence of numerous bacterial proteins present in the control material may indicate their protective role, while the presence of bacterial proteins detected only in the material taken from the placentas of the study group may indicate their potentially pathogenic nature. This phenomenon is probably important in the development of the immune system in early life, and the placental microbiota and its metabolites may have great potential in the screening, prevention, diagnosis and treatment of FGR.

The Activity of Chelidonium majus L. Latex and Its Components on HPV Reveal Insights into the Antiviral Molecular Mechanism.

Yellow-orange latex of Chelidonium majus L. has been used in folk medicine as a therapeutic agent against warts and other visible symptoms of human papillomavirus (HPV) infections for centuries. The observed antiviral and antitumor properties of C. majus latex are often attributed to alkaloids contained therein, but recent studies indicate that latex proteins may also play an important role in its pharmacological activities. Therefore, the aim of the study was to investigate the effect of the crude C. majus latex and its protein and alkaloid-rich fractions on different stages of the HPV replication cycle. The results showed that the latex components, such as alkaloids and proteins, decrease HPV infectivity and inhibit the expression of viral oncogenes (E6, E7) on mRNA and protein levels. However, the crude latex and its fractions do not affect the stability of structural proteins in HPV pseudovirions and they do not inhibit the virus from attaching to the cell surface. In addition, the protein fraction causes increased TNFα secretion, which may indicate the induction of an inflammatory response. These findings indicate that the antiviral properties of C. majus latex arise both from alkaloids and proteins contained therein, acting on different stages of the viral replication cycle.

Functional Studies of Plant Latex as a Rich Source of Bioactive Compounds: Focus on Proteins and Alkaloids.

Latex, a sticky emulsion produced by specialized cells called laticifers, is a crucial part of a plant's defense system against herbivory and pathogens. It consists of a broad spectrum of active compounds, which are beneficial not only for plants, but for human health as well, enough to mention the use of morphine or codeine from poppy latex. Here, we reviewed latex's general role in plant physiology and the significance of particular compounds (alkaloids and proteins) to its defense system with the example of Chelidonium majus L. from the poppy family. We further attempt to present latex chemicals used so far in medicine and then focus on functional studies of proteins and other compounds with potential pharmacological activities using modern techniques such as CRISPR/Cas9 gene editing. Despite the centuries-old tradition of using latex-bearing plants in therapies, there are still a lot of promising molecules waiting to be explored.

Combined Protein and Alkaloid Research of Chelidonium majus Latex Reveals CmMLP1 Accompanied by Alkaloids with Cytotoxic Potential to Human Cervical Carcinoma Cells.

Chelidonium majus L. is a latex-bearing plant used in traditional folk medicine to treat human papillomavirus (HPV)-caused warts, papillae, and condylomas. Its latex and extracts are rich in many low-molecular compounds and proteins, but there is little or no information on their potential interaction. We describe the isolation and identification of a novel major latex protein (CmMLP1) composed of 147 amino acids and present a model of its structure containing a conserved hydrophobic cavity with high affinity to berberine, 8-hydroxycheleritrine, and dihydroberberine. CmMLP1 and the accompanying three alkaloids were present in the eluted chromatographic fractions of latex. They decreased in vitro viability of human cervical cancer cells (HPV-negative and HPV-positive). We combined, for the first time, research on macromolecular and low-molecular-weight compounds of latex-bearing plants in contrast to other studies that investigated proteins and alkaloids separately. The observed interaction between latex protein and alkaloids may influence our knowledge on plant defense. The proposed toolbox may help in further understanding of plant disease resistance and in pharmacological research.

Antiviral activity of berberine.

Plants are a rich source of new antiviral, pharmacologically active agents. The naturally occurring plant alkaloid berberine (BBR) is one of the phytochemicals with a broad range of biological activity, including anticancer, anti-inflammatory and antiviral activity. BBR targets different steps in the viral life cycle and is thus a good candidate for use in novel antiviral drugs and therapies. It has been shown that BBR reduces virus replication and targets specific interactions between the virus and its host. BBR intercalates into DNA and inhibits DNA synthesis and reverse transcriptase activity. It inhibits replication of herpes simplex virus (HSV), human cytomegalovirus (HCMV), human papillomavirus (HPV), and human immunodeficiency virus (HIV). This isoquinoline alkaloid has the ability to regulate the MEK-ERK, AMPK/mTOR, and NF-κB signaling pathways, which are necessary for viral replication. Furthermore, it has been reported that BBR supports the host immune response, thus leading to viral clearance. In this short review, we focus on the most recent studies on the antiviral properties of berberine and its derivatives, which might be promising agents to be considered in future studies in the fight against the current pandemic SARS-CoV-2, the virus that causes COVID-19.

Oncogenic viruses and cancer / Wirusy onkogenne a nowotwory.

Oncogenic viruses (oncoviruses) are implicated in approximately 12% of all human cancers. Currently, the viruses known to cause human cancer are: Hepatitis B and C viruses (HBV and HCV), Human Papillomaviruses (HPV), Merkel Cell Polyomavirus (MCV), Human Herpesvirus-8 (HHV-8), Epstein-Barr Virus (EBV) and Human T-cell lymphotropic virus-1 (HTLV-1). However, oncoviruses are not complete carcinogens, need additional factors andisplay different roles in transformation. Oncoviruses can directly disrupt important regulatory cell genes by inserting virus genom into the DNA of the host cell. They also contain their own genes that damage the regulation of the cell. Some viruses have v-onc that cause disregulation of cellular processes and can lead to cancerous growth.

Antiviral compounds isolated from plants / Przeciwwirusowe zwiazki izolowane z roslin.

Viruses are intracellular pathogens which utilize a number of host metabolic processes for virus replication in addition to proteins which are encoded for virus itself. Therefore, an effective antiviral drug must interfere with virus encoded proteins without affecting any cellular metabolic processes. Unfortunately, many antiviral drugs that have an inhibitory effect on virus replication, also have an inhibitory effect on molecular processes in infected, as well as uninfected, cells. There is currently no approved remedy for many viruses. Plants represent a large potential source of antiviral agents, such as: alkaloids, flavonoids, phenolic acids, phenylpropanoids, lignins, terpenoids, quinine, tannins, thiophenes, polyacetylenes or proteins. Some of them possess broad spectrum of antiviral activity.

Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses.

Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine.

Combination of transcriptomic and proteomic approaches helps to unravel the protein composition of Chelidonium majus L. milky sap.

MAIN CONCLUSION: A novel annotated Chelidonium majus L. transcriptome database composed of 23,004 unique coding sequences allowed to significantly improve the sensitivity of proteomic C. majus assessments, which showed novel defense-related proteins characteristic to its latex. To date, the composition of Chelidonium majus L. milky sap and biosynthesis of its components are poorly characterized. We, therefore, performed de novo sequencing and assembly of C. majus transcriptome using Illumina technology. Approximately, 119 Mb of raw sequence data was obtained. Assembly resulted in 107,088 contigs, with N50 of 1913 bp and N90 of 450 bp. Among 34,965 unique coding sequences (CDS), 23,004 obtained CDS database served as a basis for further proteomic analyses. The database was then used for the identification of proteins from C. majus milky sap, and whole plant extracts analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) approach. Of about 334 different putative proteins were identified in C. majus milky sap and 1155 in C. majus whole plant extract. The quantitative comparative analysis confirmed that C. majus latex contains proteins connected with response to stress conditions and generation of precursor metabolites and energy. Notable proteins characteristic to latex include major latex protein (MLP, presumably belonging to Bet v1-like superfamily), polyphenol oxidase (PPO, which could be responsible for browning of the sap after exposure to air), and enzymes responsible for anthocyanidin, phenylpropanoid, and alkaloid biosynthesis.

Viral and other cell-penetrating peptides as vectors of therapeutic agents in medicine.

Efficient delivery of heterologous molecules for treatment of cells is a great challenge in modern medicine and pharmacology. Cell-penetrating peptides (CPPs) may improve efficient delivery of a wide range of macromolecular cargos, including plasmid DNA, small interfering RNA, drugs, nanoparticulate pharmaceutical carriers, and anticancer drugs. In this paper, we present the history of CPPs' discovery with special attention drawn to sequences of viral origin. We also describe different CPP families with regard to their physicochemical properties and numerous mechanisms of CPP cell uptake by direct penetration and endocytotic pathways. A detailed description is focused on formation of carrier-cargo complexes, which are needed for practical use of CPPs in medicine and biotechnology. Examples of successful application of CPPs in treatment of human diseases are also presented, including decreased tumor growth and induction of cancer cell death. Finally, we review modern design approaches to novel CPPs and prediction of their activity. To sum up, the current review presents a thorough and up-to-date knowledge of CPPs and may be a valuable source of information for researchers in pharmacology designing new therapeutic agents.

Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera).

We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole-larval preparations, permitting studies of the very small whole-larval preparations. Protein preparations were analyzed using FITC-casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0-4.0). Cysteine protease activity increased at acidic pH and in the presence of ß-mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe(2+) , Ca(2+) , Mg(2+) , Zn(2+) , and K(+) (10 mM). Fe(2+) and Zn(2+) significantly decreased enzyme activity at all pH values, Ca(2+) and Mg(2+) at pH 6.2 and Mg(2+) at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N-p-tosyl-l-phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, Nα -tosyl-l-lysine chloromethyl ketone hydrochloride, N-p-tosyl-l-phenylalanine chloromethyl ketone, trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin-like) proteases and metalloproteases act in cereal leaf beetle digestion.

Identification of a Coding Sequence and Structure Modeling of a Glycine-Rich RNA-Binding Protein (CmGRP1) from Chelidonium majus L.

The family of glycine-rich plant proteins (GRPs) is a large and complex group of proteins that share, as a common feature, the presence of glycine-rich domains arranged in (Gly)n-X repeats that are suggested to be involved in protein-protein interactions, RNA binding, and nucleolar targeting. These proteins are implicated in several independent physiological processes. Some are components of cell walls of many higher plants, while others are involved in molecular responses to environmental stress, and mediated by post-transcriptional regulatory mechanisms. The goals of this study are to identify the coding sequence of a novel glycine-rich RNA-binding protein from Chelidonium majus and to propose its structural model. DNA fragments obtained using degenerate PCR primers showed high sequence identities with glycine-rich RNA-binding protein coding sequences from different plant species. A 439-bp nucleotide sequence is identified coding for a novel polypeptide composed of 146 amino acids, designated as CmGRP1 (C. majus glycine-rich protein 1), with a calculated MW of 14,931 Da (NCBI GenBank accession no. HM173636). Using NCBI CDD and GeneSilico MetaServer, a single conserved domain, the RNA recognition motif (RRM), was detected in CmGRP1. The C-terminal region of CmGRP1 is a glycine-rich motif (GGGGxxGxGGGxxG), and it is predicted to be disordered. Based on a 1fxl crystal structure, a 3D model of CmGRP1 is proposed. CmGRP1 can be classified as a class IVa plant GRP, implicated to play a role in plant defense.

Herbgenomics meets Papaveraceae: a promising -omics perspective on medicinal plant research.

Herbal medicines were widely used in ancient and modern societies as remedies for human ailments. Notably, the Papaveraceae family includes well-known species, such as Papaver somniferum and Chelidonium majus, which possess medicinal properties due to their latex content. Latex-bearing plants are a rich source of diverse bioactive compounds, with applications ranging from narcotics to analgesics and relaxants. With the advent of high-throughput technologies and advancements in sequencing tools, an opportunity exists to bridge the knowledge gap between the genetic information of herbs and the regulatory networks underlying their medicinal activities. This emerging discipline, known as herbgenomics, combines genomic information with other -omics studies to unravel the genetic foundations, including essential gene functions and secondary metabolite biosynthesis pathways. Furthermore, exploring the genomes of various medicinal plants enables the utilization of modern genetic manipulation techniques, such as Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR/Cas9) or RNA interference. This technological revolution has facilitated systematic studies of model herbs, targeted breeding of medicinal plants, the establishment of gene banks and the adoption of synthetic biology approaches. In this article, we provide a comprehensive overview of the recent advances in genomic, transcriptomic, proteomic and metabolomic research on species within the Papaveraceae family. Additionally, it briefly explores the potential applications and key opportunities offered by the -omics perspective in the pharmaceutical industry and the agrobiotechnology field.

Tap the sap - investigation of latex-bearing plants in the search of potential anticancer biopharmaceuticals.

Latex-bearing plants have been in the research spotlight for the past couple of decades. Since ancient times their extracts have been used in folk medicine to treat various illnesses. Currently they serve as promising candidates for cancer treatment. Up to date there have been several in vitro and in vivo studies related to the topic of cytotoxicity and anticancer activity of extracts from latex-bearing plants towards various cell types. The number of clinical studies still remains scarce, however, over the years the number is systematically increasing. To the best of our knowledge, the scientific community is still lacking in a recent review summarizing the research on the topic of cytotoxicity and anticancer activity of latex-bearing plant extracts. Therefore, the aim of this paper is to review the current knowledge on in vitro and in vivo studies, which focus on the cytotoxicity and anticancer activities of latex-bearing plants. The vast majority of the studies are in vitro, however, the interest in this topic has resulted in the substantial growth of the number of in vivo studies, leading to a promising number of plant species whose latex can potentially be tested in clinical trials. The paper is divided into sections, each of them focuses on specific latex-bearing plant family representatives and their potential anticancer activity, which in some instances is comparable to that induced by commonly used therapeutics currently available on the market. The cytotoxic effect of the plant's crude latex, its fractions or isolated compounds, is analyzed, along with a study of cell apoptosis, chromatin condensation, DNA damage, changes in gene regulation and morphology changes, which can be observed in cell post plant extract addition. The in vivo studies go beyond the molecular level by showing significant reduction of the tumor growth and volume in animal models. Additionally, we present data regarding plant-mediated biosynthesis of nanoparticles, which is regarded as a new branch in plant latex research. It is solely based on the green-synthesis approach, which presents an interesting alternative to chemical-based nanoparticle synthesis. We have analyzed the cytotoxic effect of these particles on cells. Data regarding the cytotoxicity of such particles raises their potential to be involved in the design of novel cancer therapies, which further underlines the significance of latex-bearing plants in biotechnology. Throughout the course of this review, we concluded that plant latex is a rich source of many compounds, which can be further investigated and applied in the design of anticancer pharmaceuticals. The molecules, to which this cytotoxic effect can be attributed, include alkaloids, flavonoids, tannins, terpenoids, proteases, nucleases and many novel compounds, which still remain to be characterized. They have been studied extensively in both in vitro and in vivo studies, which provide an excellent starting point for their rapid transfer to clinical studies in the near future. The comprehensive study of molecules from latex-bearing plants can result in finding a promising alternative to several pharmaceuticals on the market and help unravel the molecular mode of action of latex-based preparations.

Placental proteome in late­onset of fetal growth restriction.

Fetal growth restriction (FGR) occurs when the fetus does not reach its genetically programmed intrauterine potential for growth and affects ~5­10% of pregnancies. This condition is one of the leading causes of perinatal mortality and morbidity associated with obstetric and neonatal complications. Placental dysfunction in FGR causes an impairment in the transfer of nutrients and oxygen from the mother to the developing fetus. Maternal adaptations to placental insufficiency may also play a role in the pathophysiology of FGR. The present study aimed to compare the proteome of the placentas of 18 women with the physiological course of pregnancy and eutrophic fetus [estimated fetal weight (EFW) >10th percentile; control group] and 18 women with late FGR (EFW <10th percentile) diagnosed after 32 weeks of pregnancy, according to the Delphi consensus (study group). The U. Mann­Whitney test was used to compare two independent groups. The R. Spearman correlation coefficient significance test was used to assess the existence of a relationship between the analyzed measurable parameters. P<0.05 was considered to indicate a statistically significant difference. The tests showed the presence of 356 different proteins which were responsible for the regulation of gene transcription control, inhibiting the activity of proteolytic enzymes, regulation of trophoblast proliferation and angiogenesis and inflammatory response. In the FGR placental proteome, other detected proteins were mostly involved in response to oxidative stress, cellular oxidation and detoxication, apoptosis, hemostatic and catabolic processes, energy transduction protein interactions, cell proliferation, differentiation and intracellular signaling. The present study used chromatographic mass­spectrometry to compare the placental proteome profiles in pregnancies complicated by late­onset FGR and normal pregnancy. Comparative analysis of proteomes from normal and FGR placentas showed significant differences. Further research is needed to clarify maternal and fetal adaptations to FGR.

Characterization and expression of a novel thaumatin-like protein (CcTLP1) from papaveraceous plant Corydalis cava.

Thaumatin-like proteins (TLPs, osmotins) form a protein family which shares a significant sequence homology to the sweet-tasting thaumatin from the plant Thaumatococcus daniellii. TLPs are not sweet-tasting and are involved in response to biotic stresses and developmental processes. Recently it has been shown using a proteomic approach that the tuber extract from Corydalis cava (Papaveraceae) contains a TLP protein. The aim of this work was to characterize the structure and expression of TLP from C. cava tubers. The results obtained using a PCR approach with degenerate primers demonstrated a coding sequence of a novel protein, named CcTLP1. It consists of 225 aa, has a predicted molecular weight of 24.2 kDa (NCBI GenBank accession no. KJ513303) and has 16 strictly conserved cysteine residues, which form 8 disulfide bridges and stabilize the 3D structure. CcTLP1 may be classified into class IX of plant TLPs. The highest CcTLP1 expression levels were shown by qPCR in the stem of the plant compared to other organs and in the medium-size plants compared to other growth phases. The results confirm that CcTLP1 is expressed during plant growth and development until flowering, with a possible defensive function against different stress conditions.

Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells.

BACKGROUND: Corydalis cava Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group Chelidonium majus L. (Greater Celandine), belong to the family Papaveraceae. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The Corydalis sp. tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in Corydalis cava tubers. METHODS: Nucleolytic proteins were isolated from the tubers of C. cava by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS). RESULTS: The studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml) - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR) proteins. CONCLUSIONS: The cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most probably, represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines.

Protoberberine compounds extracted from Chelidonium majus L. as novel natural photosensitizers for cancer therapy.

BACKGROUND: It has been shown that secondary metabolites occur in Chelidonium majus L. (C. majus) crude extract and milky sap (alkaloids such as berberine, coptisine, chelidonine, chelerythrine, sanguinarine, and protopine) are biologically active compounds with a wide spectrum of pharmacological functions. Berberine, an isoquinoline alkaloid extracted from plants, possesses a wide range of biological activities, including inhibition of growth of a variety of cancer cell lines. PURPOSE AND STUDY DESIGN: In the present study, we investigated the potential anticancer effect of a protoberberine alkaloidal fraction (BBR-F) isolated from the medicinal plant C. majus on HeLa and C33A cervical cancer cells after light irradiation (PDT treatment). METHODS: BBR-F was prepared from an ethanolic extract of stems of C. majus. Identification of alkaloidal compounds was performed using high-performance liquid chromatography - mass spectrometry (HPLC/ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. BBR-F was then biologically evaluated for its anticancer properties. Cytotoxic activity after PDT treatment and without light irradiation (dark cytotoxicity) was determined by colorimetric WST-1 assay. The impact of the protoberberine alkaloidal fraction on the morphology and function of the cells was assessed by fluorescence and confocal microscopy as well as by flow cytometric analysis. To investigate the proinflammatory effect of the extracted natural BBR-F, nitric oxide concentration was determined using the Griess method. RESULTS: An effective reduction in HeLa and C33A cell viability was observed after PDT treatment of BBR-F treated cells. Furthermore, microscopic analysis identified various morphological changes in the studied cells that occurred during apoptosis. Apoptosis of HeLa and C33A cells was also characterized by biochemical changes in cell membrane composition, activation of intracellular caspases, disruption of the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) generation. CONCLUSION: Our results strongly suggest that the components of the natural plant protoberberine fraction (BBR-F) extracted from C. majus may represent promising novel photosensitive agents and can be applied in cancer photodynamic therapy as natural photosensitizers.