The effect of the glycolipoprotein extract (G‐90) from earthworm Eisenia foetida on the wound healing process in alloxan‐induced diabetic rats

Diabetes is now regarded as a major public health problem. The number of patients is estimated to increase to over 439 million cases by 2030. One of the major health clinical problems in patients with diabetes patients is impaired wound healing. Diabetic foot ulcer is a major complication of diabetes mellitus in 12 to 25% of patients, which increases the risk of damage in the limbs or amputation. The earthworm Eisenia foetida glycolipoprotein (as known G‐90) is a blend of macromolecules with some biological properties including mitogenicity, anticoagulation, fibrinolysis, bacteriostatic and antioxidatiaon. Given the biological properties of G‐90, this study was conducted to investigate the effect of extract obtained from the homogenate of Eisenia foetida (G‐90) on the wound healing process in alloxan‐induced diabetic rats. The results of the present study revealed that treatment by using G‐90 can speed up the wound healing process, which is exactly similar to the effect of D‐panthenol treatment in rats. These findings also demonstrated that G‐90 treatment decreases the risk of infection in the wound site compared with D‐panthenol treatment. In addition, histological analysis indicated that a better extracellular matrix formation with increased fibroblast proliferation, neovascularization, collagen synthesis and early epithelial layer formation was observed in G‐90 treated group. Therefore, the G‐90 could be considered as a new wound healing agent introducing promising therapeutic approaches in both human and veterinary medicine. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

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.     

The chemical synthesis and binding affinity to the EGF receptor of the EGF-like domain of heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family of growth factors, was isolated from the conditioned medium of macrophage-like cells. To investigate the effect of N- and C-terminal residues of the EGF-like domain of HB-EGF in the binding affinity to the EGF receptor on A431 cell. We synthesized HB-EGF(44-86) corresponding to the EGF-like domain of HB-EGF and its N- or C-terminal truncated peptides. Thermolytic digestion demonstrated three disulfide bond pairings of the EGF-like domain in HB-EGF is consistent with that of human-EGF and human-TGF-alpha. HB-EGF(44-86) showed high binding affinity to EGF-receptor, like human-EGF. The truncation of the C-terminal Leu86 residue from HB-EGF(44-86), HB-EGF(45-86) or HB-EGF(46-86) caused a drastic reduction in the binding affinity to the EGF receptor. These results suggest that the EGF-like domain of HB-EGF plays an important role in the binding to the EGF receptor, and its C-terminal Leu86 residue is necessary for binding with the EGF-receptor. In addition, the deletion of the two N-terminal residues (Asp44-Pro45) from HB-EGF(44-86) caused a 10-fold decrease in relative binding affinity to the EGF receptor. This indicates that the two N-terminal residues of the EGF-like domain of HB-EGF are necessary for its optimal binding affinity to the EGF receptor.     

Nuclear translocation of cell-surface receptors: lessons from fibroblast growth factor

The nuclear localization of a number of growth factors, cytokine ligands and their receptors has been reported in various cell lines and tissues. These include members of the fibroblast growth factor (FGF), epidermal growth factor and growth hormone families. Accordingly, a number of nuclear functions have begun to emerge for these protein families. The demonstration of functional interactions of these proteins with the nuclear import machinery has further supported their functions as nuclear signal transducers. Here, we review the membrane- trafficking machinery and pathways demonstrated to regulate this cell surface to nucleus-trafficking event and highlight the many remaining unanswered questions. We focus on the FGF family, which is providing many of the clues as to the process of this unusual phenomenon.     

Presence and localization of epidermal growth factor (EGF)- and EGF-receptor-like immunoreactivity in Tetrahymena

The presence of EGF- and EGF-receptor-like immunoreactivity in Tetrahymena was studied with the help of FITC-labelled monoclonal antibodies, using flow cytometry and confocal microscopy. Tetrahymena has endogeneous EGF and treatment with it significantly increases the hormone content of the cells. The hormone is diffusely localized, particularly in the cytopharynx, where it forms a structure larger than 10 microm. EGF-receptors are also demonstrable, particularly in the cortical region in connection with cilia, and EGF treatment significantly enhances cortical fluorescence. The relationships between these observations and literary data on the effects of EGF in Tetrahymena are discussed.     

PLAC1 expression increases during trophoblast differentiation: evidence for regulatory interactions with the fibroblast growth factor-7 (FGF-7) axis

PLAC1 is a recently described, trophoblast-specific gene that localizes to a region of the X-chromosome important in placental development. Immunohistochemical analysis demonstrated that PLAC1 polypeptide localizes to the differentiated syncytiotrophoblast throughout gestation (8-41 weeks) as well as a small population of villous cytotrophoblasts. Consistent with these observations, quantitative RT-PCR demonstrated that PLAC1 mRNA increases more than 300-fold during cytotrophoblast differentiation in culture to form syncytiotrophoblasts. Agents known to be relevant to trophoblast differentiation were then tested for the ability to influence PLAC1 expression. Fibroblast growth factor-7 (FGF-7), also known as keratinocyte growth factor (KGF), stimulated PLAC1 mRNA expression approximately two-fold in the BeWo(b30) trophoblast cell line. FGF-7 stimulation was significantly inhibited by PD-98059 and wortmannin suggesting mediation via MAP kinase and PI-3 kinase-dependent signaling pathways. Interestingly, epidermal growth factor (EGF) treatment of trophoblasts had no effect on PLAC1 expression alone, but potentiated the effect of FGF-7, suggesting the presence of a regulatory interaction of the two growth factors. FGF-7 and its receptor, FGFR-2b, exhibited spatial overlap with PLAC1 suggesting these regulatory interactions are physiologically relevant during gestation. These data demonstrate PLAC1 expression is upregulated during trophoblast differentiation, localizing primarily to the differentiated syncytiotrophoblast. Furthermore PLAC1 expression is specifically regulated by peptide growth factors relevant to trophoblast differentiation.     

Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats

When rat serum was subjected to gel filtration on a Sephadex G-200 column, a factor, "hepatotropin", that promoted hepatocyte growth in primary culture was separated. Its Mr was about 150 KD and it was an anionic protein that was unstable on acid- and heat-treatments. Hepatotropin was purified 20-fold further by affinity chromatography on heparin-Sepharose CL-6B. The purified hepatotropin was effective at 20 micrograms/ml and maximally effective at 120 micrograms/ml, and its effect was additive with that of insulin plus epidermal growth factor. In rats after partial hepatectomy, the hepatotropin activity in the serum increased time-dependently reaching a maximum of about 5-fold the initial level 24 h after the operation. Various known growth factors, such as fibroblast growth factor, platelet derived growth factor, somatomedin, thrombin and transferrin, did not stimulate DNA synthesis in cultured hepatocytes. These results suggest that hepatotropin is a new growth factor.     

Further purification of a fibroblast growth factor-like factor from chick embryo extract by heparin-affinity chromatography

Summary A mitogenic factor which promotes quail myoblast proliferation has been purified some 10⁵-fold from chick embryo extract by a combination of cation-exchange chromatography and heparin-affinity chromatography. The factor is eluted from heparin-Sepharose with 2M NaCl and is a single-chain polypeptide with an apparent molecular weight of 15000 to 17000. It is active at subnanogram level in triggering the proliferation and thereby delaying temporarily fusion of myoblasts. It also stimulates the proliferation of quail fibroblasts in a similar effective concentration range. For both myoblasts and fibroblasts the dose-response to the factor is quantitatively and qualitatively comparable with that of bovine pituitary fibroblast growth factor. These observations strongly suggest that the factor very probably corresponds to chicken fibroblast growth factor or to a closely related molecule(s) and that it is possibly involved in the regulation of myogenesis. This work was partly supported by a grant from the National Center of Neurology and Psychiatry (NCNP grant 86-01) of the Ministry of Health and Welfare, Japan.     

Immunoreactive fibroblast growth factor (FGF) in a transplantable chondrosarcoma: inhibition of tumor growth by antibodies to FGF

Using a radioimmunoassay for bovine pituitary fibroblast growth factor (FGF), we have established the presence of the immunoreactive mitogen in extracts of a transplantable mouse chondrosarcoma. Both neutral and acidic extracts of the tumor contain an immunoreactive FGF (ir-FGF) that cross-reacts in a parallel and dose-dependent fashion in the radioimmunoassay. The ir-FGF is retained on heparin-Sepharose affinity columns and can be detected in the same molecular weight forms as rat pituitary FGF. Mice (C57/Bl) inoculated with the tumor (10 mg, im) show a decreased rate of tumor growth when passively immunized with the antiserum to FGF. The results establish the presence of FGF in this tumor and implicate its role in the etiology of its development.     

Short form of the heparin-binding EGF-like growth factor: Communication II

The structure of the green monkey Chlorocebus aethiops heparin-binding EGF-like growth factor (HB-EGF) gene was compared with that of the corresponding human gene. Exon 3a, characteristic of the short form of HB-EGF (SF-HB-EGF), was mapped between exons 3 and 4, approximately 700 bp away from the latter. In several human and simian cell lines, most of the SF-HB-EGF mRNA proved to lack exons 4 and 5, specific to the HB-EGF mRNA. In contrast to the HB-EGF mRNA, the SF-HB-EGF mRNA occurred predominantly in the P, rather than L, form, which codes for a protein with a different propeptide structure. Labeled SF-HB-EGF competed with HB-EGF and EGF for binding to the surface of A431 cells, suggesting its interaction with the specific EGF receptor. The results indicate that SF-HB-EGF plays a specific role in cell signaling.     

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

Hormonal interactions in Tetrahymena: effect of hormones on levels of epidermal growth factor (EGF)

Tetrahymena pyriformiswas treated with insulin, histamine or serotonin for 30 min and epidermal growth factor (EGF) level was studied inside the cells using specific antibodies and flow cytometry as well as confocal microscopy. The EGF concentration was highly significantly elevated after hormone treatment, regardless of the hormone used. EGF was localized mainly in the cortical region (mucocysts) and in vesicles and this localization did not differ in untreated and treated cells. The results call attention to the possibility of interactions between hormones at unicellular level and points to the presence of a hormonal system in Tetrahymena that includes receptors, hormones and signal transduction pathways as well as hormonal interactions. This could be the basis of further evolution to the hormonal system of multicellulars.     

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.     

Improved production of recombinant fibroblast growth factor 7 (FGF7/KGF) from bacteria in high magnesium chloride

Because of specificity for both heparin/heparan sulfate and the receptor complex on epithelial cells relative to other fibroblast growth factor (FGF) homologues, there is considerable interest in clinical and commercial applications of FGF7 (also called keratinocyte growth factor or KGF) that require large quantities at reasonable cost. Production of recombinant FGF7 from bacteria suffers from lower yields and recovery relative to FGF1 and FGF2. Fusion of FGF7 at the N-terminus with glutathione-S-transferase (GST) followed by removal of GST by proteolysis while bound to natural ligand heparin improved the intrinsically low yields from Escherichia coli hosts to 3.2 mg per liter per OD(600), which was still only 10% of that for FGF1. Yield of the GST-FGF7 fusion product was improved to about 17 mg per liter per OD(600) in strain BL21(DE3)pLysS by inclusion of 10-100mM magnesium chloride (MgCl(2)) in the culture medium. This improved by about five times the yields of fully active 54ser-FGF7 after proteolytic excision of the GST portion from GST-FGF7 immobilized on heparin-Sepharose. This simple enhancement improves the cost-effectiveness of production of recombinant FGF7 in bacteria for clinical and commercial applications.     

Unravelling the insulin-like growth factor I-mediated photoprotection of the skin

Chronic exposure of human skin to solar ultraviolet radiation (UVR) induces a range of biological reactions which may directly or indirectly lead to the development of skin cancer. In order to overcome these damaging effects of UVR and to reduce photodamage, the skin’s endogenous defence system functions in concert with the various exogenous photoprotectors. Growth factors, particularly insulin-like growth factor-I (IGF-I), produced within the body as a result of cellular interaction in response to UVR demonstrates photoprotective properties in human skin. This review summarises the impact of UVR-induced photolesions on human skin, discusses various endogenous as well as exogenous approaches of photoprotection described to date and explains how IGF-I mediates UVR photoprotective responses at the cellular and mitochondrial level. Further, we describe the current interventions using growth factors and propose how the knowledge of the IGF-I photoprotection signalling cascades may direct the development of improved UVR protection and remedial strategies.     

恶性转化的成纤维细胞表皮生长因子受体的研究

本文用受体的放射性配基结合分析方法观察了C_3H小鼠胚胎成纤维细胞C_3H_(10)T1/2 CL8(简称NC_3H_(10))和~3H-TdR恶性转化的C_3H_(10)T1/2CL8(简称TC_3H_(10))的表皮生长因子受体(EGFR)。结果表明细胞恶性转化前后的EGFR都存在高亲和力和低亲和力两种结合位点,细胞恶性转化后能结合表皮生长因子的EGFR结合位点减少,Western blotting和受体的亲和交联分析表明EGFR的分子量为170kD,是单链多肽。     

Ligand-dependent and -independent interactions with the transforming growth factor type II and I receptor subunits reside in the aminoterminal portion of the ectodomain of the type III subunit

Summary The type III receptor for transforming growth factor beta (TGFβ), which exhibits no kinase activity, binds TGFβ1 and TGFβ2 and is involved in assembly and activity of the multi-subunit TGFβ signal transduction complex. Recently we showed that TGFβ receptor type III (TβRIII) can participate in a complex composed of the dimeric TGFβ ligand and a type III, II, and I receptor subunit. The interaction of the TβRIII subunit with TβRII is TGFβ-dependent, whereas interaction with TβRI is TGFβ-independent. Here we use coexpression of the three types of TGFβ receptors in baculoviral-infected insect cells to determine which parts of the unglycosylated TβRIII receptor participate in the binding of TGFβ, the TGFβ-dependent interaction with TβRII and the TGFβ-independent interaction with TβRI. The results suggest that the first 500 amino acid residues in the aminoterminal portion of TβRIII exhibit all three properties.