Potent Mitogenic Activity Research Articles

Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy.

Introduction: Human basic fibroblast growth factor (hbFGF) is a highly valuable multifunctional protein that plays a crucial role in various biological processes. In this study, we aim to accomplish the scaling-up production of mature hbFGF (146aa) by implementing a high cell-density fermentation and purification process on a 500-L scale, thereby satisfying the escalating demands for both experimental research and clinical applications. Methods: The hbFGF DNA fragment was cloned into a mpET-3c vector containing a kanamycin resistance gene and then inserted into Escherichia coli BL21 (DE3) plysS strain. To optimize the yield of hbFGF protein, various fermentation parameters were systematically optimized using BOX-Behnken design and further validated in large-scale fermentation (500-L). Additionally, a three-step purification protocol involving CM-Sepharose, heparin affinity, and SP-Sepharose column chromatography was developed to separate and purify the hbFGF protein. Isoelectric focusing electrophoresis, MALDI-TOF/MS analysis, amino acid sequencing, CD spectroscopy, and Western blotting were performed to authenticate its identity. The biological efficacy of purified hbFGF was evaluated using an MTT assay as well as in a diabetic deep second-degree scald model. Results: The engineered strain was successfully constructed, exhibiting high expression of hbFGF and excellent stability. Under the optimized fermentation conditions, an impressive bacterial yield of 46.8 ± 0.3g/L culture with an expression level of hbFGF reaching 28.2% ± 0.2% was achieved in 500-L scale fermentation. Subsequently, during pilot-scale purification, the final yield of purified hbFGF protein was 114.6 ± 5.9mg/L culture with RP-HPLC, SEC-HPLC, and SDS-PAGE purity exceeding 98%. The properties of purified hbFGF including its molecular weight, isoelectric point (pI), amino sequence, and secondary structure were found to be consistent with theoretical values. Furthermore, the purified hbFGF exhibited potent mitogenic activity with a specific value of 1.05 ± 0.94 × 106AU/mg and significantly enhanced wound healing in a deep second-degree scald wound diabetic rat model. Conclusion: This study successfully established a stable and efficient large-scale production process of hbFGF, providing a solid foundation for future industrial production.

Pleiotrophin-Loaded Mesoporous Silica Nanoparticles as a Possible Treatment for Osteoporosis.

Osteoporosis is the most common type of bone disease. Conventional treatments are based on the use of antiresorptive drugs and/or anabolic agents. However, these treatments have certain limitations, such as a lack of bioavailability or toxicity in non-specific tissues. In this regard, pleiotrophin (PTN) is a protein with potent mitogenic, angiogenic, and chemotactic activity, with implications in tissue repair. On the other hand, mesoporous silica nanoparticles (MSNs) have proven to be an effective inorganic drug-delivery system for biomedical applications. In addition, the surface anchoring of cationic polymers, such as polyethylenimine (PEI), allows for greater cell internalization, increasing treatment efficacy. In order to load and release the PTN to improve its effectiveness, MSNs were successfully internalized in MC3T3-E1 mouse pre-osteoblastic cells and human mesenchymal stem cells. PTN-loaded MSNs significantly increased the viability, mineralization, and gene expression of alkaline phosphatase and Runx2 in comparison with the PTN alone in both cell lines, evidencing its positive effect on osteogenesis and osteoblast differentiation. This proof of concept demonstrates that MSN can take up and release PTN, developing a potent osteogenic and differentiating action in vitro in the absence of an osteogenic differentiation-promoting medium, presenting itself as a possible treatment to improve bone-regeneration and osteoporosis scenarios.

The relationship between clinical parameters and hepatocyte growth factor/c-Met levels in the sperm of infertile males

Objective: Hepatocyte growth factor is a pleiotropic cytokine with potent mitogenic activity in many organs and tissues. The receptor for hepatocyte growth factor is c-Met, a transmembrane glycoprotein with tyrosine kinase activity. We investigated the relationships between hepatocyte growth factor and c-Met expression levels on sperm motility and male infertility. Methods: The study was designed as a prospective cohort, single-center clinical trial and was conducted between March 2015 and June 2016. The control group consisted of 31 healthy male volunteers with children, while the study group consisted of 61 men who were diagnosed with male infertility after presenting to the In Vitro Fertilization Unit of our Institute. All participants in the study were aged 18–60 years. Sperm samples were taken from each participant and divided in two. The fresh sample was examined immediately for expression of c-Met, while the other was stored frozen and evaluated later for hepatocyte growth factor. Primary outcome measures were the levels of hepatocyte growth factor and c-Met expression, while secondary outcome measures were the correlation of these levels with sperm count, morphology and motility. Results: The c-Met expression level in the control group (26.53 ± 3.50, 19.43–32.73) was significantly lower than in the infertile group (27.95 ± 2.86, 21.58–33.07) (p = 0.039). HGF expression was also significantly lower in the control group (3.25 ± 1.76, 0.83–8.25) than in the infertile group (4.87 ± 4.11, 1.01–21.58) (p = 0.043). In the overall group, c-Met expression showed a positive correlation with sperm motility percentage and a negative correlation with sperm count and sperm morphology percentage. Conclusions: The c-Met receptor and its ligand, hepatocyte growth factor, appear to be fundamental regulators of spermatogenesis, sperm motility and fertilization capacity.

Large-Scale Preparation of Highly Stable Recombinant Human Acidic Fibroblast Growth Factor in Escherichia coli BL21(DE3) plysS Strain.

In this study, the optimum human aFGF gene encoding haFGF135 was cloned in pET3c and transferred to Escherichia coli BL21(DE3) plysS. To enhance the yield of fermentation and the expression level of the target protein, the fermentation parameters, including temperature, pH, dissolved oxygen, glucose concentration, ammonium chloride concentration, induction time, and inducer (IPTG) concentration, were optimized. The optimized fermentation parameters were used in large-scale fermentation (30 L). Ion-exchange and heparin-affinity column chromatography techniques were used for separation and purification of rhaFGF135 protein. HPLC, isoelectric focusing electrophoresis, and mass spectrometry were used to detect the purity, isoelectric point, and molecular weight and peptide map of rhaFGF135 protein, respectively. Mitogenic activity of rhaFGF135 protein was detected in NIH-3T3 cells and a full-thickness injury wound diabetic rat model. The production and expression level of rhaFGF135 in the 30-L scale fermentation reached 80.4 ± 2.7 g/L culture and 37.8% ± 1.8%, respectively. The RP-HPLC and SDS-PAGE purity of the final rhaFGF135 product almost reached 100%, and the final pure protein yield was 158.6 ± 6.8 mg/L culture. Finally, the cell and animal experiments showed that rhaFGF135 retained a potent mitogenic activity. The large-scale process of rhaFGF135 production reported herein is relatively stable and time-saving, and thus, it can be used as an efficient and economic strategy for the synthesis of rhaFGF135 at the industrial level.

Purification and characterization of a heterodimeric mycelial lectin from Penicillium proteolyticum with potent mitogenic activity

In the present study, Penicillium proteolyticum lectin was purified by DEAE-Sepharose chromatography followed by Sephadex G-100 gel filtration chromatography. A sequence of ion-exchange and gel filtration chromatography resulted in 52.30 fold purified lectin with a high yield of 84.21%. The purified P. proteolyticum lectin is a glycoprotein with 2.83% linked carbohydrates. A single band in Native-PAGE, whereas two bands (25.1 kDa and 22.9 kDa) in SDS-PAGE confirmed the heterodimeric nature of the purified lectin. The apparent molecular weight (48 kDa) of P. proteolyticum lectin was further confirmed by gel filtration analysis. Heterodimeric P. proteolyticum lectin was stable within a pH range of 6.5-7.5 and upto a temperature of 30 °C. The lectin activity was strongly inhibited by complex glycoproteins and denaturants. Metal ions are not required for agglutination activity of purified lectin. The lectin showed significant mitogenic response towards mice splenocytes. It exhibited highest mitogenic activity at a concentration of 50 μg/mL. This is a first report on characteristics of purified P. proteolyticum lectin having potent mitogenic potential.

Purification and characterization of a mitogenic lectin from Penicillium duclauxii

Lectins are proteins/glycoproteins of non-immune origin which interact specifically and non-covalently with carbohydrate moieties on the cell surface. In this study, a lectin was purified from Penicillium duclauxii by ion-exchange chromatography on DEAE-Sepharose and gel filtration chromatography on a Sephadex G-100 column. An overall recovery of 94.11% and 60-fold purification was achieved. The purified lectin had a molecular weight of 54.9 kDa and was found to be heterogeneous as revealed by double band of sub-units with molecular mass of 21.13 kDa and 33.26 kDa, under reducing conditions. It is a glycoprotein with carbohydrate content of 3.95%. Lectin induced haemagglutination of erythrocytes was inhibited strongly by glycoproteins such as bovine submaxillary mucin, porcine stomach mucin and fetuin. The maximum haemagglutinating activity of P. duclauxii lectin was maintained after incubation at a temperature and pH range of 20–35 °C and 6.0–8.0, respectively. The haemagglutinating activity of P. duclauxii lectin was unaffected by EDTA and various metal ions. The purified P. duclauxii lectin exhibited maximum mitogenic activity towards mouse splenocytes at a concentration of 75 μg/mL. This manuscript reports a novel lectin from P. duclauxii with potent mitogenic activity towards mouse splenocytes.

Abstract B17: Xenospheres: a comprehensive patient-derived in vitro model to study response and resistance to targeted therapies in metastatic colorectal cancer

Abstract Although EGFR targeted therapy has improved the survival outcome of patients with metastatic colorectal cancer, lack of response or emergence of secondary resistance frequently occurs as result of genetic alterations that hyperactivate the RAS pathway or by activation of compensatory pathways by signals from the tumor microenvironmet that can also induce a stem-like phenotype. From patient-derived xenografts (“xenopatients”) of colorectal cancer liver metastases, we generated primary sphere cultures (“xenospheres”) that display the properties of cancer stem-like cells. Indeed, these cells can long-term self-propagate in vitro, and form phenocopies of original patient tumors in vivo (“spheropatients”), maintaining the same response to anti-EGFR therapy. We obtained a panel of xenospheres derived from more than 40 different xenopatients, all genetically characterized for the presence of mutations in genes that are predictive for response to EGFR targeted therapy. The panel included xenospheres harboring mutations in RAS genes (Rasmut), or recently identified rare predictors of resistance: ERBB2 amplification/mutation, MET amplification, EGFR mutation in the extracellular domain, and IGF2 overexpression. Moreover, we conducted an extensive exome-sequencing analysis of 10 xenospheres, for which the exome data of the matched xenopatients were available, thus allowing a complete and robust comparison. Xenospheres displayed a good genetic stability and a remarkable correspondence with xenopatients, in both the mutational and gene expression profiles. While RASmut xenospheres displayed self-sustained proliferative ability, those lacking mutations affecting the RAS pathway (RASwt) were strongly dependent of exogenous growth factors. We thus assessed the response of all RASwt xenospheres to ligands of the EGF family (TGFA, EREG, AREG, HB-EGF, NRG1), or bFGF, or HGF, testing both proliferation and response to EGFR inhibition (cetuximab). We found that TGFA and HB-EGF induced proliferation and resistance to cetuximab with almost the same potency of EGF, while EREG and AREG had a weak mitogenic activity and did not protect against cetuximab treatment, accordingly with observations in patients and xenopatients. The different proliferative activity (and protection against cetuximab) can be at least in part explained by the effect on receptor stabilization and exposure to the cell membrane exerted by different ligands. Interestingly, we found that, among all EGF family ligands, NRG1 (binding ERBB3/4) had the most potent mitogenic activity and protection against cetuximab treatment. NRG1 was able to replace EGF in sustaining xenosphere propagation, without altering the global gene expression profile, implying that it could sustain cancer-stem like cell properties similarly to EGF. To study the role of NRG1 in vivo we generated both an autocrine and a paracrine mode, by inducing its expression infecting either RASwt xenospheres or a cell line of murine fibroblasts. NRG1-expressing RASwt xenospheres became growth factor independent, resistant to cetuximab but sensitive to lapatinib (a dual EGFR/ERBB2 small molecule). Similar results were obtained using the conditioned medium of NRG1-expressing murine fibroblasts. By injecting parental and modified RASwt xenospheres into immunocompromised mice, we found that NRG1-expressing xenospheres had a strongly increased tumor onset, and that these tumors were resistant to cetuximab but sensitive to lapatinib treatment, suggesting its possible use in the clinical practice of RASwt patients. We conclude that xenospheres are a robust patient-derived in vitro model of colorectal cancer-stem like cells, amenable to study molecular mechanisms of response or resistance to targeted therapies. Citation Format: Paolo Luraghi, Viola Bigatto, Gigliola Reato, Elia Cipriano, Francesco Sassi, Claudio Isella, Andrea Bertotti, Livio Trusolino, Paolo Maria Comoglio, Carla Boccaccio. Xenospheres: a comprehensive patient-derived in vitro model to study response and resistance to targeted therapies in metastatic colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B17.

T19: The immune and vascular components of microenvironment in prognosis of the endometrioid endometrial adenocarcinoma

Background Endometrioid endometrial adenocarcimoma is the most frequent gynecologic malignancy in the western world. It is well known that inflammation plays a key role in the initiation and development of cancer as well as promotes both specific and innate immune response. There is no information on the status of the local anti-tumor cellular immunity in endometrioid adenocarcinoma of the corpus uteri microenvironment in the Russian and English language medical scientific literature. Various studies have shown that high intratumor microvessel density in endometrial cancer is associated with advanced stage, increased risk of recurrent disease and with poor prognosis. Vascular endothelial growth factor (VEGF) is a glycoprotein with potent mitogenic activity. It has been demonstrated to be a key component in the promotion of angiogenesis. Materials and methods There were singlet 24 cases of endometrioid adenocarcinoma with retrospectively known good (survival) and poor (death) outcomes. All cases were immunohistochemically stained for CD3, CD20, CD57, CD68, S100, CD34 and VEGF, expression of was evaluated by IHC-profiler. The number and the area of vessels was count in “hot spot” zones and calculated per 1 mm. For compare of the characteristics of signs was used comparison of two independent samples – U-Mann–Whitney test. The level of statistical significance was accepted by p Results T-lymphocytes cells were situated predominantly in desmoplastic stroma of the tumor. CD3 expression was significant higher in the group of good outcome (p = 0.00042), B-lymphocytes cells were predominantly in epithelial component of tumor. CD20 was expressed in endometriod adenocarcinoma with good and poor outcome without statistically significant difference (p = 0.976). NK-lymphocytes were predominantly in peritumoral stroma. CD57 expression was statistically significant higher in the group of good outcome (p = 0.000307). Tumor infiltrating macrophages can were frequent in tumor stroma than in tumor islets. CD68 expression cells number was higher in the group with good outcome (p = 0.000219). Dendric cells were predominantly in epithelial component of tumor. S100+ cells number did not have any difference (p = 0.105). Inside the tumor stroma, among pseudo-glands or solid beaches there was a positive reaction for anti-CD34 of numerous microvessels together with groups of endothelial cells (“hot spot”). The number of intratumoral vessels in patients with poor outcome was 141.9 ± 24.7 vessels/mm2, in good outcome patients – 93.8 ± 28.7 vessels/mm2. The number of the intratumoral vessels in this groups was with significant statistical different (p = 0.0014). The area of intratumoral vessels in good outcome cases was 2799.7 ± 535.7 μm2, poor outcome cases – 5455,2 ± 448,8 μm2. It was significant statistical different (p = 0.000014). VEGF was expressed in the cytoplasm of normal endometrial cells, neoplastic ones, endothelial cells and stroma. VEGF represents a mitogen for endothelial cells that induces the formation of lumen’s vessels and also increases vascular permeability and protein extravasation. Expression of VEGF by cancer cells had significant statistical difference in this groups of patients (p = 0.0411). Conclusion There was a significant reduction in T, NK-cells and macrophages from patients with poor outcome indicates the development of local immune deficiency that could certainly plays an important role in tumor progression endometrioid carcinoma. Our study demonstrates that the number and the area of vessels, as part of angiogenesis, represent an important prognostic factor in outcome of endometrioid endometrial adenocarcinoma. VEGF expression also is an angiogenic factor which plays a great role in the prognosis of this tumor.

Purification and characterization of a mycelial mucin specific lectin from Aspergillus panamensis with potent mitogenic and antibacterial activity

A lectin was isolated from Aspergillus panamensis mycelia using single-step affinity chromatography on porcine stomach mucin coupled Sepharose 4B. A purification fold of 19.2 with 62.5% yield was achieved by this method. Gel filtration chromatography showed the native molecular mass of 78kDa and the subunit molecular mass of 39kDa was confirmed by 10% SDS-PAGE and MALDI-TOF analysis. Isoelectric focussing of native A. panamensis lectin revealed it to be an acidic protein with pI 4.5. Carbohydrate content of purified lectin was estimated to be 4.95%. All database search of N-terminal amino acid sequences revealed no homology of the purified lectin with previously reported microbial, viral and animal lectins except 29kDa subunit of heterodimeric Vigna sesquipedalis lectin. The lectin from this fungal source is a homodimer. Lectin was fairly thermostable with pH optima of 6.5–8.0 and had no requirement of divalent metal cations for its activity. Maximum mitogenic activity toward mouse spleen cells was exhibited at 100μg/mL concentration of purified lectin. Results of the antimicrobial assay showed that the lectin had potent antibacterial activity against Bacillus cereus, Staphylococcous aureus, Pseudomonas aeruginosa and Escherichia coli.

MicroRNA-145 directly targets the insulin-like growth factor receptor I in human bladder cancer cells

The insulin-like growth factor receptor I (IGF-IR) is a proto-oncogene with potent mitogenic and antiapoptotic activities. It has been reported that expression of IGF-IR is up-regulated in bladder cancer. Here, we assessed whether microRNA-145 (miR-145) regulates IGF-IR expression in bladder cancer. In our study, miR-145 was shown to directly target IGF-IR 3′-untranslated region (UTR) in human bladder cancer cells. Small interfering RNA (siRNA)- and miR-145-mediated IGF-IR knockdown experiments revealed that miR-145 promotes cell apoptosis, and suppresses cell proliferation and migration through suppression of IGF-IR expression. Taken together, our data suggest that miR-145 may inhibit bladder cancer initiation by affecting IGF-IR signaling.

진균류 균사체의 고체발효 커피생두로부터 조제한 원두커피의 생리활성

커피의 생리활성을 증진시키기 위해 고체발효를 이용하여 인도네시아산 Mandheling 커피생두에 3종의 버섯 균사체(Phellinus linteus, PL; Hericium erinaceum, HE; Ganoderma lucidum, GL) 및 2종의 홍국균 균사체(Monascus purpureus, MP; Monascus ruber, MR)를 배양하였다. 균사체-고체발효 커피생두를 로스팅하여 얻은 원두커피는 decoction법에 의한 열수추출물과 reflux에 의한 에탄올추출물로 조제하였는데, 열수추출물(HW, 수율 17.7～25.3%)은 에탄올추출물(EE, 9.5～12.2%)보다 더 높은 수율을 나타내었다. 2종의 홍국균 균사체-고체발효 커피생두로부터 조제된 원두커피 열수추출물(MP-CB-HW, MR-CB-HW)은 비발효 원두커피 또는 3종 버섯 균사체-고체발효 원두커피 열수추출물보다 높은 총 폴리페놀 및 플라보노이드 함량과 DPPH 자유라디칼 소거능을 나타내었다. 또한, 홍국균 균사체-고체발효 원두커피 중에서도 MR-CB-HW는 가장 높은 마크로파지 활성과 마이토젠 활성을 나타내었다(CB-HW의 1.32배와 1.40배). MP-CB-EE와 MR-CB-EE는 100 μg/mL 의 시료농도에서 세포에 대한 독성을 나타내지 않으면서도 LPS로 유도된 RAW 264.7 세포의 산화질소(NO)의 생성을 효과적으로 억제하였다(LPS 처리군의 38.6과 37.0%). 한편, 홍국균 균사체를 이용한 고체발효는 카페인 함량에 영향을 주지 않으면서 클로로겐산을 유의적으로 증가시켰다(76.21～76.73 μg/mL). 결론적으로 원두커피의 생리활성은 M. purpureus 또는 M. ruber와 커피생두의 고체발효에 의해서 증진되었으며 이러한 결과는 홍국균-고체발효 원두커피가 기능성 커피음료의 소재로 이용될 가능성을 제시하는 것으로 사료된다.

Metformin and Cancer in Type 2 Diabetes

Metformin and Cancer in Type 2 Diabetes

Structural characterization and antitumor and mitogenic activity of a lectin from the gill of bighead carp (Aristichthys nobilis)

In this study, we investigated the gross structure, secondary structure, and antitumor and mitogenic activity of GANL, a lectin from the gill of bighead carp (Aristichthys nobilis). We used infrared spectroscopy, β-elimination, and circular dichroism spectroscopy to determine the structure of GANL. We measured antiproliferation activity against six human tumor cell lines and mitogenic activity against murine splenocytes using the MTT assay. Based on infrared spectroscopy and β-elimination, we conclude that GANL is a glycoprotein. The protein and carbohydrate moieties are joined by O-glycosidic linkage. A circular dichroism spectroscopic analysis revealed that the secondary structure of GANL consists of α-helices (34.8 %), β-sheets (12.1 %), β-turns (24.5 %), and unordered structures (33.0 %). GANL exerted potent antitumor activity against the HeLa cell line (IC(50) = 11.86 μg/mL) and a mitogenic effect on murine splenocytes in the MTT assay. GANL, a lectin that is isolated from the gills of bighead carp, is a glycoprotein with potent antitumor and mitogenic activity.

Antivascular endothelial growth factor agents for neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of severe visual loss and blindness over the age of 50 in developed countries. Vascular endothelial growth factor (VEGF) is considered as a critical molecule in the pathogenesis of choroidal neovascularization (CNV), which characterizes the neovascular AMD. Anti-VEGF agents are considered the most promising way of effectively inhibition of the neovascular AMD process. VEGF is a heparin-binding glycoprotein with potent angiogenic, mitogenic and vascular permeability-enhancing activities specific for endothelial cells. Two anti-VEGF agents have been approved by the US Food and Drug Administration (FDA) for the treatment of neovascular AMD. Pegaptanib sodium, which is an aptamer and ranibizumab, which is a monoclonal antibody fragment. Another humanized monoclonal antibody is currently off-label used, bevacizumab. This paper aims to discuss in details the effectiveness, the efficacy and safety of these three anti-VEGF agents. New anti-VEGF compounds which are recently investigated for their clinical usage (VEGF-trap, small interfering RNA) are also discussed for their promising outcomes.

Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities

Letter to the Glyco-Forum: Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities

Anti-VEGF Compounds in the Treatment of Neovascular Age Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness among elderly patients in developed countries. Although the pathogenesis of AMD is still largely unknown, it is now well known that vascular endothelial growth factor (VEGF) plays a pivotal role in the growth of the abnormal blood vessels (i.e. choroidal neovascularization, CNV) which characterizes the "wet form" of this ocular disease. Therefore, inhibiting VEGF has turned out to be a good way of more effectively controlling neovascular AMD. VEGF is a heparin-binding glycoprotein with potent angiogenic, mitogenic and vascular permeability-enhancing activities specific for endothelial cells. Currently two anti-VEGF compounds have been approved by the US Food and Drug Administration (FDA) for the treatment of neovascular AMD: pegaptanib and ranibizumab. Off-label usage of bevacizumab, an anti-VEGF agent similar to ranibizumab, has also become fairly common. The substantial improvement of visual acuity noticed in patients treated with ranibizumab has made this drug the gold standard for AMD therapy. However, as with many new therapies, there are unresolved issues, including safety, cost, and dosing frequency. This review describes in details the properties and efficacy of the three anti-VEGF agents in use in clinical practice. Promising emerging anti-VEGF strategies (VEGF-trap, small interfering RNA, tyrosine kinase inhibitors) which aim to improve outcomes, safety and treatment burden through novel mechanisms of action are also discussed.

Mitogenic Activity of CEL-I, anN-Acetylgalactosamine (GalNAc)-Specific C-Type Lectin, Isolated from the Marine InvertebrateCucumaria echinata(Holothuroidea)

An N-acetylgalactosamine (GalNAc)-specific Ca(2+)-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 microg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 microg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 microg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.

Purification and characterization of a mucin-binding mycelial lectin from Aspergillus nidulans with potent mitogenic activity

Mucin-specific lectin from mycelium of Aspergillus nidulans was purified using anion exchange and gel filtration chromatographic techniques with an overall recovery of 32% and 21.97-fold purification. The purified lectin migrated as a single band in SDS–PAGE with an apparent molecular mass of 34 kDa. Carbohydrate analysis revealed that it is a glycoprotein with total sugar content of 2.54%. Optimal agglutination was observed when serially diluted lectin was incubated with human type O erythrocyte suspension at pH 7.0–8.0 and temperature 20–30°C. Lectin was found to be completely stable within pH 5.0–8.0 and temperature at or below 40°C. Demetallization by extensive dialysis against EDTA did not alter its haemagglutination activity. Lectin activity was reduced to half after 24 h incubation with urea and thiourea, with no such effect of guanidine HCl. The lectin showed potent mitogenic response towards mouse splenocytes, attaining a maximum at 200 μg/ml as compared to untreated control cells. Mitogenic lectins are invaluable tools to assess the functioning of immune cells. None of the microfungal lectin has yet been investigated for mitogenic activity. This is the first report on mitogenic activity of lectin from Aspergillus sp.

The Human IGF1R IRES likely operates through a Shine–Dalgarno‐like interaction with the G961 loop (E‐site) of the 18S rRNA and is kinetically modulated by a naturally polymorphic polyU loop

IGF1R is a proto-oncogene with potent mitogenic and antiapoptotic activities, and its expression must be tightly regulated to maintain normal cellular and tissue homeostasis. We previously demonstrated that translation of the human IGF1R mRNA is controlled by an internal ribosome entry site (IRES), and delimited the core functional IRES to a 90-nucleotide segment of the 5'-untranslated region positioned immediately upstream of the initiation codon. Here we have analyzed the sequence elements that contribute to the function of the core IRES. The Stem2/Loop2 sequence of the IRES exhibits near-perfect Watson-Crick complementarity to the G961 loop (helix 23b) of the 18S rRNA, which is positioned within the E-site on the platform of the 40S ribosomal subunit. Mutations that disrupt this complementarity have a negative impact on regulatory protein binding and dramatically decrease IRES activity, suggesting that the IGF1R IRES may recruit the 40S ribosome by a eukaryotic equivalent of the Shine-Dalgarno (mRNA-rRNA base-pairing) interaction. The homopolymeric Loop3 sequence of the IRES modulates accessibility and limits the rate of translation initiation mediated through the IRES. Two functionally distinct allelic forms of the Loop3 poly(U)-tract are prevalent in the human population, and it is conceivable that germ-line or somatic variations in this sequence could predispose individuals to development of malignancy, or provide a selectable growth advantage for tumor cells.

A Novel Lectin with Antiproliferative and HIV-1 Reverse Transcriptase Inhibitory Activities from Dried Fruiting Bodies of the Monkey Head MushroomHericium erinaceum

A lectin designated as Hericium erinaceum agglutinin (HEA) was isolated from dried fruiting bodies of the mushroom Hericium erinaceum with a chromatographic procedure which entailed DEAE-cellulose, CM-cellulose, Q-Sepharose, and FPLC Superdex 75. Its molecular mass was estimated to be 51 kDa and its N-terminal amino acid sequences was distinctly different from those of other isolated mushroom lectins. The hemagglutinating activity of HEA was inhibited at the minimum concentration of 12.5 mM by inulin. The lectin was stable at pH 1.9–12.1 and at temperatures up to 70°C, but was inhibited by Hg2+, Cu2+, and Fe3+ ions. The lectin exhibited potent mitogenic activity toward mouse splenocytes, and demonstrated antiproliferative activity toward hepatoma (HepG2) and breast cancer (MCF7) cells with an IC50 of 56.1 μM and 76.5 μM, respectively. It manifested HIV-1 reverse transcriptase inhibitory activity with an IC50 of 31.7 μM. The lectin exhibited potent mitogenic activity toward murine splenocytes but was devoid of antifungal activity.