Influences of earthworm extract G-90 on haematological and haemostatic parameters in Wistar rats.

OBJECTIVES: A balance between fibrinolysis and coagulation is crucial for maintaining haemostasis. A haemostatic disorder leads to various physiological changes and causes different diseases. Glycolipoprotein mixture (G-90), prepared from the tissue homogenate of the earthworm Eisenia foetida, was examined in vivo, in an animal study (conducted on Wistar rats) in order to determine its fibrinolytic and anticoagulation activity. MATERIALS AND METHODS: The influence of G-90 treatment on haematological and homeostatic parameters was monitored as well. RESULTS: Statistical analysis has shown the most pronounced effect of G-90 to be exerted on bleeding and coagulation times; the effects in reference were proven to be statistically significant (p = 0.03 and 0.005, respectively). A statistically significant effect of G-90 was also seen with thrombin time (p = 0.02) and plasminogen level (p = 0.004). CONCLUSION: The results have shown the influence of G-90 on blood coagulation to be very similar to that of heparin, a known anticoagulant. Thus G-90 could be considered as a new thrombolytic agent of use in veterinary and human medicine.

The delivery of biologically active (therapeutic) peptides and proteins into cells.

Biologically active peptides and proteins have a great potential to act as targeted drug therapies in the treatment of a variety of diseases, including cancer. However, their use in vivo is limited by their low stability and cell permeability. Thus, it is necessary to develop efficient and safe peptide/protein delivery systems that can overcome these problems and increase a therapy's bioavailability. The search for promising vectors has led to the use of compounds called cell-penetrating peptides or protein transduction domains. The cell-penetrating peptides, as effective transporter, are utilized to enhance uptake of various biologically active peptide/protein cargos upon fusion or attachment to its sequences. Cell-penetrating peptides have been the subject of investigation of many researchers, however this review only focuses on the arginine-rich and amphipathic carriers and their potential therapeutic use.

Tissue extract from Eisenia foetida as a wound-healing agent.

BACKGROUND AND OBJECTIVES: This study was aimed at demonstrating the influence of Eisenia foetida earthworm extract (G-90) on wound healing in an animal model. Medicinal properties of earthworms have been long known, especially in Eastern countries. MATERIALS AND METHODS: Four groups of Wister rats (36 in each group) were wounded under anaesthesia; the skin was surgically incised in a circular manner (the circular incision in reference measuring 2 cm in diameter) and afterwards daily treated for 24 days in a following manner: Group 0--the control group deprived from any treatment whatsoever; Group 1--treated with physiological saline solution; Group 2--treated with Panthenol D and serving as a positive control, and Group 3--treated with G-90 (10 ng/ml). The animals were sacrificed on days 3, 6, 9, 12, 18 and 24 post wounding and the diameter of wound was measured by virtue of photometric method. The excised wounds were routinely fixed and embedded into paraffin section and dully stained for histopathological analysis. The presence of microorganisms on the wounds was assessed as well. RESULTS: The best antibacterial wound shielding was achieved with G-90 treatment. Besides antibacterial shielding, the wounds treated with G-90 were also protected from inflammation. G-90 was shown to shorten the inflammatory, and accelerate the proliferative and the maturation phase, stimulating thereby the regeneration of an injured epidermis. Statistical analysis revealed G-90 (p=0.018) to be superior over other treatments. CONCLUSIONS: Thus, Eisenia foetida earthworm extract (G90) might be considered as a new wound-healing agent suitable for use in both veterinary and human medicine practice.

Alternative sources of fibrinolytic, anticoagulative, antimicrobial and anticancer molecules.

The medicinal properties of earthworms in various remedies date back to 1340 A.D. and have been extended to other countries and cultures. Assays of tissue homogenates of earthworm (Eisenia foetida) have revealed a glycolipoprotein mixture referred to as G-90 that is composed of macromolecules with medical and pharmaceutical applications. There are several functions attributed to G-90: possession of several growth factors that: stimulate proliferation in cell cultures, contain an insulin like growth factor (IGF like), an immunoglobulin like growth factor (IgFG-like), possess two serine peptidases with a tyrosine code and epidermal growth factor (EGF). In contrast, G-90 exerts strong fibrinolytic and anticoagulative activity capable of lysing fibrin clots. Actions of these two properties are dependent upon concentration. Anti-coagulative activity also depends upon the kind of anticoagulants (G-90, PI, PII). G-90 can also act as antioxidant, exert antimicrobial activities in vitro and in vivo. The bacteriostatic effect is significantly greater for non-pathogenic species. Finally G-90 also participates in tissue regeneration and wound healing. Taken together, components of earthworms could be tested in certain clinical trials.

Stimulation of growth factor synthesis in skin wounds using tissue extract (G-90) from the earthworm Eissenia foetida.

Growth factors are biologically-active mediators that bind to specific receptors on target cells and regulate genes involved in cell growth, wound healing and regeneration. In the case of wound healing, a proper wound dressing is needed to cover the wound area, protect the damaged tissue, and if possible to activate cell proliferation and stimulate the healing process. In this study we examined the efficacy of a glycolipoprotein tissue homogenate extract from Eisenia foetida (G-90) to activate signal transduction pathways, leading to wound healing. We measured the activation of EGF and FGF in healthy skin, in wounds with physiological healing and in wounds treated with G-90. The activation of EGF and FGF was measured during the first 24 h of wound healing under both physiological conditions and treatment with G-90. In both cases an increased concentration of EGF and FGF was observed 6 h after wounding. In comparison with healthy skin, the concentration of EGF increased 10-fold and FGF five-fold in wounds treated with G-90 (10 ng ml(-1)). Healing in physiological conditions resulted in a two-fold increase of EGF and 1.5-fold of FGF.

Synthesis, crystal structure and antiproliferative evaluation of some new substituted benzothiazoles and styrylbenzothiazoles.

The multistep synthesis of a series of new substituted-benzothiazoles as hydrochloride or quaternary salts is described. 6-Amidino substituted 2-aminobenzothiazoles (5, 6), N-methyl-2-(4-cyanostyryl)benzothiazolium iodide (8), cyano-substituted-2-styrylbenzothiazoles (9-11) and amidino and bis-amidino-substituted 2-styrylbenzothiazoles (12-17) were prepared. The crystal structure of amidino derivative (6) was determined by single crystal X-ray analysis. All new prepared compounds were tested on the cytostatic activities against malignant cell lines: (SW620, colon carcinoma; Hep2, laryngeal carcinoma; HBL, melanoma; HeLa, cervical carcinoma and WI38, human normal fibroblasts). The compounds exerted a different inhibitory effect, depended on concentration and type of the cells. The best inhibitory effect was achieved with compounds (12-15), with slight differences among them. All of them inhibited the growth of examined tumor cell lines and also normal fibroblasts. Other examined compounds exhibited a moderate inhibitory effect, depending on type of the cells. Majority of them inhibited the growth of HeLa cells and WI38.

Synthesis and antitumor evaluation of some new substituted amidino-benzimidazolyl-furyl-phenyl-acrylates and naphtho[2,1-b]furan-carboxylates.

The multistep synthesis of a series of substituted amidino-benzimidazolyl-furyl-phenyl-acrylic acid's esters and substituted amidino-benzimidazolyl-naphtho[2,1-b]furan-carboxylic acid's esters is described starting from corresponding 3-(2-furyl)-2-phenyl-acrylic acids. The new compounds were tested on the cytostatic activities against malignant cell lines: pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa), laryngeal carcinoma (Hep2), colon carcinoma (HT 29), melanoma (HBL), and human fibroblasts cell line (WI38). All compounds inhibited the proliferation of tumor cell lines. Inhibitory effect of examined compounds depended on concentration, but without significant difference among the type of tumor cells. The compounds 2 and 5 exerted very low inhibitory effect on the growth of human fibroblasts. Unsubstituted derivative 8 has not inhibited any tested cell lines.

Effect of earthworm (G-90) extract on formation and lysis of clots originated from venous blood of dogs with cardiopathies and with malignant tumors.

The stability of homeostasis is important to keep a balance between coagulation and fibrinolysis. A disorder of homeostasis leads to different physiological changes and causes different diseases such as cardiopathies and malignant tumors. Cardiopathies is characterized by a hypercoagulation. In the malignant tumors, besides the hypercoagulation due to plasminogen activators (PA) formed inside the tumor, a disorder of homeostasis leads also to acceleration of the fibrinolysis. The variety of internal and external factors in both cases determine the deviation of time for the clots formation, as well as the lyses of blood and fibrin clots. In this study the venous blood as well as the blood and the fibrin clots, derived from healthy dogs, the dogs with cardiopathies and with malignant tumors, were examined for the time of coagulation and fibrinolysis by adding different substances. In these experiments we used a glycolipoprotein extract from earthworm tissue homogenate (G-90) and the proteolytic enzymes P I and P II, isolated from G-90. The efficacy of the tested substances was comparable with the clinically administered anticoagulants. The most significant differences in clotting time among the three tested groups of dogs were obtained by application of the original G-90. The results suggest a possibility that G-90, along with the fibrinolytic enzymes and other biologically active factors, also contains a factor that decelerates the formation of clot in a specific medium, such as the blood from the dogs with malignant tumors.

Glycolipoprotein extract (G-90) from earthworm Eisenia foetida exerts some antioxidative activity.

Antioxidants protect DNA, proteins and lipids in the body from damage. These types of damages are a major contributor to aging and to degenerative diseases such as cancer, cardiovascular disease, immune-system decline, brain dysfunction and cataracts. The effect of glycolipoprotein extract of Eisenia foetida (G-90) as an antioxidant was investigated in cultured human fibroblasts and epithelial cells. After treatment of the cells with H2O2 for 4 h, G-90 completely allows the cells to recover and stimulated their growth. When the cells were incubated with G-90 48 h before the treatment with H2O2, the oxidative damage of the cells did not occur. Thus, G-90 had an apparent protective effect against the toxicity of H2O2 and stimulated the growth of the cells. Ascorbic acid, a known antioxidant, did not allow the growth of the cells to recover after damage nor did it protect them, unless it was added simultaneously with H2O2. The antioxidative activity of G-90, together with its antibacterial and mitogen activities, could be useful in the study of G-90 as a wound-healing agent.

Erythrocytic differentiation and glyceraldehyde-3-phosphate dehydrogenase expression are regulated by protein phosphorylation and cAMP in HD3 cells.

Utilisation of glucose undergoes a marked decline during erythroblastic differentiation in the chicken. Concomitantly there is a reduction in the expression of glucose transporter proteins and in the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAD). GAD activity declines, after an initial rise, while the level of GAD mRNA decreases rapidly after induction of differentiation. We have employed the temperature-sensitive chicken erythroblast cell line HD3 that differentiates to the erythrocyte phenotype at 42 degrees C in the presence of inducers (hemin and butyric acid). The role of tyrosine and serine/threonine phosphorylation pathways were evaluated with the phosphatase inhibitors sodium vanadate and okadaic acid, respectively. In the presence of phosphatase inhibitors, HD3 cells underwent differentiation and increased their synthesis of hemoglobin which is a marker protein for red blood cells differentiation. The levels of both GAD mRNA and enzymatic activity were increased by phosphatase inhibitors. The role of cAMP in differentiation was also assessed. Differentiation of HD3 cells was associated with an increase in cAMP. However the phosphodiesterase inhibitor IBMX was not a good inducer of hemoglobin synthesis but did induce GAD mRNA and enzymatic activity. Together these results suggest that multiple pathways (including serine/threonine phosphorylation, tyrosine phosphorylation and elevated cAMP) are involved in the regulation of erythroblastic differentiation, hemoglobin synthesis, GAD gene expression and GAD activity in HD3 cells.

Regulation of glucose transport in differentiating HD3 cells.

The chicken erythroblast cell line, HD3, has high glucose transport activity which is lost upon differentiation to the red cell phenotype. HD3 cells, when incubated under conditions where maturation occurs, show substantial loss of GLUT1 and GLUT3 mRNAs. To assess whether cAMP or cellular protein phosphorylation affected GLUT mRNA and protein, the HD3 cells were incubated in the presence of different phosphatase inhibitors. Treatment of HD3 cells with the phosphatase inhibitors okadaic acid, vanadate or with 3-isobutyl-1-methyl-xanthine induced glucose transport and GLUT mRNAs. This suggests that phosphorylation events enhance glucose transport and that their reduction may be involved in the decrease in glucose transport that occurs upon HD3 cells differentiation.

Novel pyrimidine and purine derivatives of L-ascorbic acid: synthesis and biological evaluation.

The novel pyrimidine derivatives 1-6 of 2,3-dibenzyl-4,5-didehydro-5, 6-dideoxy-L-ascorbic acid were synthesized by the condensation of pyrimidine bases with 5,6-diacetyl-2,3-dibenzyl-L-ascorbic acid (DDA). Both N-9 (7) and N-7 (8) regioisomers were obtained in the reaction of 6-chloropurine with 5-acetyl-6-bromo-2, 3-dibenzyl-L-ascorbic acid (ABDA), while the reaction of 6-(N-pyrrolyl)purine with ABDA afforded exclusively the N-9 isomer 9. Structures of all newly prepared compounds were deduced from the chemical shifts in (1)H and (13)C NMR spectra, as well as connectivities in 2D homo- and heteronuclear correlation spectra. An unambiguous proof of the structure and conformation of 7 was obtained by X-ray crystallographic analysis. Compounds 1-9 were found to exert cytostatic activities against malignant cell lines: pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa), laryngeal carcinoma (Hep2), murine leukemia (L1210/0), murine mammary carcinoma (FM3A), and human T-lymphocytes (Molt4/C8 and CEM/0), as well as antiviral activities against varicella-zoster virus (TK(+)VZV and TK(-)VZV) and cytomegalovirus (CMV). The compound 6 containing a trifluoromethyl-substituted uracil ring exhibited marked antitumor activity. The N-7-substituted purine regioisomer 8 had greater inhibitory effects on the murine L1210/0 and human CEM/0 cell lines than the N-9 isomer 7. Compound 9 with the 6-purine-substituted pyrrolo moiety had a more pronounced selective cytostatic activity against human (Molt4/C8 and CEM/0) cell lines than murine (L1210/0 and FM3A/0) and human (MiaPaCa2, MCF7, HeLa, and Hep2) tumor cell lines and normal fibroblasts (Hef522). The compound 6 exhibited the most potent antiviral activities against TK(+)VZV, TK(-)VZV, and CMV, albeit at concentrations that were only slightly lower than the cytotoxic concentrations.

Alteration of phosphotyrosine-containing proteins during differentiation of chicken erythroleukemia cells (HD3).

After treatment of HD3 cells with erythroid-inducing agents (hemin and butyric acid) at 42 degrees C, the profile of phosphotyrosine-containing proteins was altered. Upon induction the overall level of phosphotyrosine-containing proteins increased. To examine the role of protein phosphorylation in HD3 cells differentiation, the cells were treated with specific inhibitors. In the presence of okadaic acid, cell proliferation was arrested and accompanied by a marked increase in haemoglobin synthesis, a differentiation marker of erythroid cells. Okadaic acid caused decrease of the phosphotyrosine-containing proteins, presumably to maintain a balance between phosphorylation/dephosphorylation processes in the cells. Addition of 3-isobutyl-1-methyl-xanthine, an activator of phosphatases, caused a decrease or disappearance of almost all phosphotyrosine-containing proteins and, at the same time, prevented the erythroid differentiation of HD3 cells. Sodium orthovanadate, a specific inhibitor of phosphotyrosine phosphatase, increased the level of phosphotyrosine proteins and induced differentiation of HD3 cells. These results indicate that phosphorylation of cellular proteins is coupled with a reaction(s) which is responsible for triggering the differentiation of HD3 cells. The phosphorylation/dephosphorylation processes are associated with an early event(s) during the differentiation of HD3 cells and may not be connected to tyrosine residues.

Regulation of glyceraldehyde-3-phosphate dehydrogenase in differentiating HD3 cells.

Red blood cells usually replenish their ATP pools by glycolysis, fueled by glucose imported via the cell membrane. Mature red cells of some species (e.g. pig, chicken) have, however, been reported to show very low glucose transport. The subject of this study was the possible dependency of the level of a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAD) on glucose transporter activity during the maturation of chicken red cells. The chicken pronormoblast cell line, HD3, was used as a model system. These cells contain higher levels of GAD and glucose transporter activities than normal chicken bone marrow cells, but reduce their levels during maturation. In an attempt to assess whether the decrease in GAD activity is regulated by the glucose transport, the chicken GLUT3 expressed under the control of viral promoter was introduced into HD3 cells by retroviral infection (pDOL-cGT3). Upon cell differentiation and maturation, both cells with and without the exogenous transporter decreased GAD activity. Butyric acid did not affect the regulation of GAD activity upon differentiation. These results show that the development of chicken red cells is manifested by reduction of their GAD activity and that this is not affected by their sugar transporter activity. The very low GAD activity in embryonic chicken red cells is thus due to a loss of this activity at an early stage in their development. Because of the very low glucose transport and GAD activities in mature chicken red cells, rates of glycolysis are likely to be low and suggesting an alternative pathway for ATP production in these cells.

Fibrinolytic and anticoagulative activities from the earthworm Eisenia foetida.

Biologically active glycolipoprotein complex (G-90) isolated from whole earthworm tissue extract shows anticoagulative and fibrinolytic activities. We isolated two tyrosine like serine peptidases with molecular masses of 34 kDa (P I) and 23 kDa (P II), respectively. P I peptidase is autocatalytically degraded to P II. Both peptidases exhibit fibrinolytic and anticoagulative activities. The activity of P I is much higher. P I in concentration of 10(5) ng ml-1 of plasma shortened the physiological time of fibrin clot lysis by 54% and completely inhibited blood clotting at a concentration of 10(3) ng ml-1 of venous blood.

Effect of a 17-member azalide on tumor cell growth.

The action of the 17-member azalide 4'-demycarosyl-20-deoxo-20-(di-N-benzylamino)-8a-aza-8a-homotyl osin was examined in vitro using five different human cell lines: laryngeal carcinoma (Hep2), pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), neuroblastoma, and normal diploid fibroblast (Hef522). After exposure, the cell growth was arrested, and morphological changes occurred in a dose-dependent manner. At a concentration of 10(-4) M the azalide completely inhibited the growth of all cell lines examined and induced morphological changes such as cell shrinkage, chromatin condensation, and DNA fragmentation. These features point to apoptosis.

Loss of glucose transport in developing avian red cells.

Although red cells are generally associated with significant glucose transport and dependence on glycolysis, the mature red cells of some species (e.g. pig) show very low glucose transport. The generally low level of glucose transport in mature mammalian red cells is the result of maturational development, since it has been shown that even in red cells which have negligible glucose transport (e.g. pig red cells) the corresponding reticulocytes have significant glucose transport activity. The reticulocytes of the chicken, however, show minimal glucose transport activity. But this also is the result of maturational development, since chicken bone marrow red cells do transport glucose which diminishes upon cell maturation in vitro. The erythroblast chicken cell line, HD3, has high glucose transport activity which is lost upon induction to the red cell phenotype. Growing HD3 cells have much higher levels of transport than native chicken bone marrow cells and this is associated in part with elevation of glucose transporter (GLUT) mRNAs as a consequence of the expression of the v-erbA and v-erbB oncogenes. Both native bone marrow red cells and HD3 cells, when incubated in vitro under conditions where maturation occurs, show substantial losses of GLUT mRNA and GLUT proteins. To assess whether the inducers of maturation (hemin and butyrate) affect only the normally expressed GLUTs, chicken GLUT3 expressed from a different promoter was introduced into the HD3 cell by retroviral infection. Both the endogenous and exogenous transporters were lost upon cell differentiation and maturation, leaving a cell with low glucose transport activity. Conversely, in growing cells, butyrate had a pronounced effect on the elevation of the GLUT3 mRNA, especially on the exogenous GLUT3 mRNA, and elevated glucose transport prior to differentiation. These results are consistent with the conclusion that chicken red cell development involves a requirement to reduce glucose transport activity. The near absence of glucose transport in the embryonic chicken red cell is thus due to a loss of this transporter during early development which occurs at an earlier developmental stage in the chicken red cell than in the mammalian red cell.

Adhesins of immunoglobulin-like superfamily from earthworm Eisenia foetida.

1. From the biologically active extract (G-90) isolated from the tissue homogenate of Eisenia foetida immunoglobulin-like structures were isolated and named G-90/4. 2. G-90/4 in nanogram concentrations stimulated cell proliferation more than did the original G-90. It lyses cells in microgram concentrations. 3. G-90/4 acts as an adhesion molecule between the receptors of adjacent cells. 4. The increase in proliferative activity was accompanied by the elevation of cytoplasmic protein containing tyrosine. 5. Immunohistochemical analyses confirm immunoglobulin-like transmembrane structures in the connective and muscular tissues of E. foetida.

Expression of glyceraldehyde-3-phosphate dehydrogenase during differentiation of HD3 cells.

The chicken erythroblast cell line HD3, which is infected with a temperature-sensitive avian erythroleukemia virus, becomes committed to differentiate to an erythrocyte upon temperature shift in the presence of inducers (hemin and butyric acid). The activity of glyceraldehyde-3-phosphate dehydrogenase (GAD), a key enzyme in the glycolytic pathway, was examined. Upon induction of differentiation the following changes in glyceraldehyde-3-phosphate dehydrogenase activity and the corresponding mRNA level occurred. Twenty-four hours post-induction the glyceraldehyde-3-phosphate dehydrogenase message decreased and virtually disappeared within 48 h. Glyceraldehyde-3-phosphate dehydrogenase activity did not follow the mRNA level and increased within 48 h post-induction and then started to fall. The discrepancy between glyceraldehyde-3-phosphate dehydrogenase activity and the mRNA level is likely due to a difference in GAD protein and mRNA half-lives. The results also suggest that enzyme activity could be regulated by post-translational events. Chicken erythrocytes expressed reduced levels of glyceraldehyde-3-phosphate dehydrogenase activity. Thus the low level of GAD found in chicken erythrocytes is associated with a turn off of GAD gene expression upon induction of erythroid differentiation.