Isolation of Echimidine and Its C-7 Isomers from Echium plantagineum L. and Their Hepatotoxic Effect on Rat Hepatocytes.

Echimidine is the main pyrrolizidine alkaloid of Echium plantagineum L., a plant domesticated in many countries. Because of echimidine's toxicity, this alkaloid has become a target of the European Food Safety Authority regulations, especially in regard to honey contamination. In this study, we determined by NMR spectroscopy that the main HPLC peak purified from zinc reduced plant extract with an MS [M + H]+ signal at m/z 398 corresponding to echimidine (1), and in fact also represents an isomeric echihumiline (2). A third isomer present in the smallest amount and barely resolved by HPLC from co-eluting (1) and (2) was identified as hydroxymyoscorpine (3). Before the zinc reduction, alkaloids (1) and (2) were present mostly (90%) in the form of an N-oxide, which formed a single peak in HPLC. This is the first report of finding echihumiline and hydroxymyoscorpine in E. plantagineum. Retroanalysis of our samples of E. plantagineum collected in New Zealand, Argentina and the USA confirmed similar co-occurrence of the three isomeric alkaloids. In rat hepatocyte primary culture cells, the alkaloids at 3 to 300 µg/mL caused concentration-dependent inhibition of hepatocyte viability with mean IC50 values ranging from 9.26 to 14.14 µg/mL. Our discovery revealed that under standard HPLC acidic conditions, echimidine co-elutes with its isomers, echihumiline and to a lesser degree with hydroxymyoscorpine, obscuring real alkaloidal composition, which may have implications for human toxicity.

Health-Promoting of Polysaccharides Extracted from Ganoderma lucidum.

Medicinal mushrooms are rich sources of pharmacologically active compounds. One of the mushrooms commonly used in traditional Chinese medicine is Ganoderma lucidum (Leyss. Ex Fr.) Karst. In Asian countries it is treated as a nutraceutical, whose regular consumption provides vitality and improves health. Ganoderma lucidum is an important source of biologically active compounds. The pharmacologically active fraction of polysaccharides has antioxidant, immunomodulatory, antineurodegenerative and antidiabetic activities. In this review, we summarize the activity of Ganoderma lucidum polysaccharides (GLP).

Triterpenoid Acids as Important Antiproliferative Constituents of European Elderberry Fruits.

In Europe, both the fruits and flowers of Sambucus nigra L. have been used against cold, as well as laxative, diaphoretic, and diuretic remedies. There are also a number of commercially available food products that contain elderberry juice, puréed or dried elderberries. Recent comprehensive literature data on pharmacology and chemistry of Sambuci fructus have encouraged us to screen extracts with different polarities from this plant material against cancer cell lines. The cytotoxic activity of the ethyl acetate and aqueous acetone extracts from elderberries as well as detected triterpenoids on human colon adenocarcinoma cell line (LoVo) and human breast cancer cell line (MCF-7) was investigated by sulforhodamine B assay. Moreover, cell migration assay was conducted for triterpenoid fraction and pure compounds. Aqueous acetone extract possessed much lower IC50 value in cancer cell lines compared to ethyl acetate extract. The latter manifested high cytotoxicity against studied cell lines, suggesting that nonpolar compounds are responsible for the cytotoxic activity. Indeed, the phytochemical analysis revealed that ursolic and oleanolic acids are the main triterpenoids in the mentioned extract of which ursolic acid showed the highest activity with IC50 values of 10.7 µg/mL on MCF-7 and 7.7 µg/mL on LoVo cells.

Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker specific for glandular epithelium and nervous tissue. Further studies should be performed to determine the structure of the tissue epitope recognized.

Cytotoxic effects of four aescin types on human colon adenocarcinoma cell lines.

Four types of aescin that are available on the pharmaceutical market, beta-aescin crystalline, beta-aescin amorphous, beta-aescin sodium and aescin polysulfate, have been analyzed for their cytotoxic effects on human colon adenocarcinoma (LoVo) and doxorubicin-resistant human colon adenocarcinoma cell lines (LoVo/Dx). Their cytotoxic activities were evaluated by sulforhodamine B (SRB) and methyl tetrazolium (MTT) assays. All four types of aescin exerted strong dose-dependent cytotoxicity to LoVo and, to a lesser degree, LoVo/Dx cell lines. The IC50 value for the LoVo/Dx cell line was higher, but still dose-dependent. Results from both assays demonstrated that p-aescin crystalline has the most cytotoxic activity toward human colon adenocarcinoma cell lines.

Enolase-like protein present on the outer membrane of Pseudomonas aeruginosa binds plasminogen.

Pseudomonas aeruginosa is one of the pathogenic bacteria which utilize binding of the host plasminogen (Plg) to promote their invasion throughout the host tissues. In the present study, we confirmed that P. aeruginosa exhibits binding affinity for human plasminogen. Furthermore, we showed that the protein detected on the cell wall of P. aeruginosa and binding human plasminogen is an enolase-like protein. The hypothesis that alpha-enolase, a cytoplasmatic glycolytic enzyme, resides also on the cell surface of the bacterium was supported by electron microscopy analysis. The plasminogen-binding activity of bacterial cell wall outer membrane enolase-like protein was examined by immunoblotting assay.

Release of an ~55kDa fragment containing the actin-binding domain of ß-spectrin by caspase-8 during FND-induced apoptosis depends on the presence of protein 4.1.

Analyses of the status of the membrane spectrin-based skeleton during fludarabine/mitoxantrone/dexamethasone-induced (FND-induced) apoptosis revealed proteolytic degradation of ß-spectrin, with the prevalent appearance of a specific fragment with a molecular weight of ~55kDa, containing the actin-binding domain (ABD). Appearance of this fragment was dependent on induction of apoptosis. In silico proteolysis of spectrin identified caspase-8 as a candidate protease responsible for the generation of this ~55kDa ABD-containing fragment. Analyses of spectrin and procaspase-8 localization during early apoptosis indicated temporary (<30-120min) submembranous colocalization of both proteins. Proteolytic release of the N-terminal ~55kDa fragment of purified spectrin by recombinant caspase-8 does not occur in normal cells, but does occur in isolated membrane, such as red blood cell ghosts, or in vitro in the presence of apoptotic cell extracts. Surprisingly, proteolysis of purified spectrin by recombinant caspase-8 resulted in the generation of the ~55kDa fragment only in the presence of purified protein 4.1. This suggests that only the appropriate spatial arrangement of the spectrin-based membrane skeleton or the appropriate conformational state of spectrin, which are both known to be induced by 4.1, can sensitize ß-spectrin to cleavage by caspase-8 at the N-terminal ABD-containing region.

Estimation of the action of three different mechlorethamine doses on biochemical parameters during experimentally induced pleuritis in rats.

Nitrogranulogen (NTG) may modify the character of inflammatory reactions. These modifications are a result of cytotoxic and mutagenic effects. NTG has high affinity to DNA and causes disorders in the synthesis of acute phase proteins (e.g., haptoglobin, transferrin, fibrinogen, and complement protein C3). Our previous studies have shown that small doses of NTG can enhance immunological defense reactions in the organism. The aim of the current studies was to determine how different NTG doses cause changes in the values of biochemical parameters in pleuritis-induced rats. The animals were randomized into five groups: Group I - control group; Group II - IP (induced pleuritis) group; Group III - NTG5 group; Group IV - NTG50 group; Group V - NTG600 group. Blood was collected from all groups of animals at 24, 48, and 72 h after the initiation of the carrageenin-induced inflammatory reaction. These investigations revealed that a dose of 5 µg NTG/kg b.w. (body weight) can change the character of the inflammation. Our studies also show that a dose of 600 µg NTG/kg b.w. causes a rapid decrease in the level of C3 at the 72 h of the experiment (after 3 applications every 24 h), which indicates a cytotoxic action of such a large NTG dose. NTG used at doses of 50 and 600 µg/kg b.w. causes the opposite metabolism of albumins and other serum proteins. Our studies show that the different doses of NTG have distinct effects on the inflammatory reaction.

[The composition, biochemical properties and toxicity of snake venoms]. / Sklad i wlasciwosci biochemiczne oraz toksycznosc jadów wezy.

2.5 million cases of snake bites are noticed in the world every year (within 100,000 is mortal). These bites occur frequently in Asia and Africa. Some reports proved the toxicity and composition changes of well-known venoms from the same snake species according to the climatic zone. Snake venom is a natural source of many biologically active substances, including those with potential therapeutic properties. These substances contain peptides, proteins, and enzymes which are divided into five subfamilies: three-finger toxins, serine protease inhibitors of the Kunitz type, phospholipases A2, serine proteases, and metalloproteases. All snake venoms are grouped depending on their mode of action. They usually cause neurotransmission disorders, cardiotoxic action, hemostasis disorders, and have central nervous system and necrotic activity.

Localization of enolase in the subfractions of a breast cancer cell line.

Enolase detected on the cell surface may be a receptor for certain ligands, especially for plasminogen. It is important for the pathogen invasiveness and in the development of a tumour. Therefore, we sought to preliminarily determine the enolase location and catalytic activity in the subfractions of MCF-7 cells. The latter was done on intact cells and in subfractions of MCF-7 cells. We identified enolase by immunoblotting. The binding of human plasminogen to enolase was performed by immunoblotting using monoclonal antibodies against plasminogen. The intact MCF-7 cells demonstrated activity of enolase. Enolase in postnuclear and perinuclear fractions is catalyticly active too. We identified the enolase protein in immunoblots of these fractions, except for the nuclear subfraction. These results provide evidence that enolase is present on the intact surface of MCF-7 cells and in post- and perinuclear fractions. The surface protein maintained catalytic activity, which suggests that its location in the plasma membrane didn't change the active centre of the enzyme.

[Amphibian skin secretions as a new source of antibiotics and biologically active substances]. / Substancje wydzielane przez gruczoly skórne plazów jako nowe zródlo antybiotyków i zwiazków biologicznie czynnych.

So far, the main sources of biologically active substances used in medicine have been plants, molds, and propolis. The obtained compounds have either therapeutic features or require additional modification. They are sometimes combined with other pharmacological substances to intensify their therapeutic effect. However, the effectiveness of many drugs has been rapidly decreasing.The overuse of antibiotics in the treatment and prophylaxis of human infections (especially in hospitals) as well as their widespread and often unjustified use in the treatment and prophylaxis of farm animal illnesses contribute to the development of a variety of resistance mechanisms by microorganisms. Because of the increasing ineffectiveness of antibiotics used so far and difficulties in obtaining new drugs, it is necessary to find new sources of these compounds, for example in animal organisms. Research has demonstrated that amphibian skin secretions are rich in a variety of active substances which have strong pharmacological properties. In these compounds we can distinguish, for example, toxins, antimicrobial peptides, opioid peptides, steroids, and alkaloids.These compounds show cytotoxic, antimicrobial, analgesic, anti-inflammatory, and even antiviral activities (including anti-HIV). These substances can be used in cell receptor studies and in transmembrane ion transport analysis. Because these compounds are secreted by skin glands,they can be easy obtained without injuring these animals. It is probable that amphibian skin constitutes a potential source of modern drugs.

[Characterization of an inflammatory response]. / Charakterystyka odczynu zapalnego.

The intensity of an inflammatory response in a tissue or an organ is dependent on the efficiency of the organism's homeostatic mechanisms, which restrict the extent of the reaction. The type of factor inducing a inflammatory response and its strength have significant influence on the dynamics of an inflammatory reaction. The prompt eradication of an inflammatory factor and its biologically adverse effects attest to the efficacious adaptive mechanisms of the organism. The inflammatory response expresses biochemical, hematological, and immunological responses at the local or systemic level.

[Receptors for advanced glycation end products and their physiological and clinical significance]. / Receptory koncowych produktów zaawansowanej glikacji - znaczenie fizjologiczne i kliniczne.

The receptor for advanced glycation end products (RAGE) is a multiligand cell-surface protein and belongs to the immunoglobulin superfamily. RAGE is expressed by different cell types, including macrophages, lymphocytes, endothelial, neuronal, and smooth muscle cells. In addition to advanced glycation end products (AGEs), RAGE binds amphoterin, S100/calgranulin, amyloid, transthyretin, and a leukocyte integrin, Mac-1. Engagement of RAGE in intracellular signaling leads to the activation of the proinflammatory transcription factor NF-kappaB to sustained cellular dysfunction and tissue destruction. In this study a pivotal role of RAGE in the progression of various diseases, i.e. diabetes, inflammation, neurodegeneration, tumors, vascular injury, atherosclerosis, and septic shock, is presented.

Distribution of beta-enolase in normal and tumor rat cells.

Enolase - a glycolytic enzyme is also expressed on the surface of eukaryotic cells such as macrophages, neutrophils, endothelial, neuronal, tumor cells. Surface enolase as plasminogen receptor plays an important role in myogenesis, tumorgenesis and angiogenesis. Determination of enolase localization in the cell lines may give rise to the elucidation of its receptor function in tumor cells. The cellular localization of the muscle-specific isoform of the enolase in normal rat cardiomyocytes (H9c2, an embryonic rat heart-derived cell line) and a rat sarcoma (R1) cell line is reported here. Immunocytochemical assays showed that this enolase isoform is freely diffused in the sarcoplasm of rat cells. The evident location of enolase molecules on the perinuclear surface is observed in immunofluorescence assays. Enolase localization on the surface of some intact normal rat cardiomyocytes was also observed. This surface protein maintains enolase catalytic activity.

[Enolase on the surface of prockaryotic and eukaryotic cells is a receptor for human plasminogen]. / Enolaza na powierzchni komórek eukariota i prokariota jako receptor plazminogenu ludzkiego.

Enolase was long considered an enzyme of the glycolytic pathway ubiquitously occurring in the cytosol of prokaryotic and eukaryotic cells. Results of extensive studies, especially those performed in the last ten years, indicate, however, that this protein is multifunctional. It plays several noncatalytic functions in various types of cells. Enolase exposed on the surface of cells may be a receptor for certain ligands. Especially interesting is its role as a receptor to human plasminogen. The enolase/plasminogen (plasmin) system is one of the mechanisms facilitating the invasiveness of pathogens in the human organism and it plays an important role in processes of myogenesis and in the development of tumor tissues. The presence of enolase on the surface of pathogenic cells invading the human organism is also a cause of antibody induction, which may be a basis for the development of certain autoimmune diseases. These questions are the subject of this review.