Human Epidermal Growth Factors Research Articles

MOLECULAR DOCKING AND INTERACTION OF COUMARIN DERIVATIVES AS INHIBITORS OF GROWTH FACTOR RECEPTORS

Objective: Cancer is the largest cause of mortality in the globe, accounting for over 10 million deaths in 2020, or roughly one in every six. Because growth factor receptors are involved in the pathophysiology of the disease in several ways, human epidermal growth factors (HER-2 and HER-3) and vascular endothelial growth factor receptors (VEGFR-2 and VEGFR-3) might be as considered therapeutic targets. Coumarins and derivatives were chosen for this study to investigate their pharmacological characteristics and therapeutic effect against the targeted proteins implicated in the pathogenesis of different cancers. Methods: In this work, 50 coumarins and their derivatives were chosen to assess their binding affinity with the targeted proteins (HER-2, HER-3, VEGFR-2, and VEGFR-3). PyRx, a virtual tool, was utilized to perform molecular docking. The investigation was carried out computationally, with data and molecular structures of the phytocompounds and proteins derived from Indian medicinal plants, phytochemistry, and therapeutics, as well as PubChem. The protein structure was validated using a variety of tools, including Protein Data Bank sum generate and BIOVIA discovery studio software. The pharmacological assessment of the ligands was carried out using ADMET filters. Results: According to the results of molecular docking, the ligands Glycycoumarin, Mutisifurocoumarin, Thunberginol A, Pervilleanine, Licocoumarin A, and Murrayacoumarin C had the lowest binding affinity toward the four targeted proteins. Conclusion: Since these compounds are effective against the growth factor receptors implicated in cancer pathogenesis, they could be a suitable candidate for cancer management and suppression. However, in vitro research is still required to support these findings.

A review of efficacy and safety of cetuximab and bevacizumab-based monoclonal antibodies in head and neck cancer.

A combination of monoclonal antibodies prescribed along with the conventional standard of care has a potential to provide significant improvement in patients suffering from head and neck cancer. This combination has also shown a significant decrease in toxicities and improved overall quality of life. Cetuximab acts by inhibiting the human epidermal growth factors as its overexpression in head and neck tumours that are responsible for treatment failure, resistance, and metastasis. Whereas, bevacizumab acts by inhibiting the vascular endothelial growth factor since its overexpression leads to induction of tumour angiogenesis. Current research has not shown any remarkable beneficial effect in disease outcomes. Thus, the addition of these monoclonal antibodies to the standard regimen for head and neck cancer can be considered a prospect that might be beneficial. Cetuximab has already been included as an option under special recommendations in recurrent/metastatic head and neck cancer by NCCN in a platinum-based regimen as well as in combination with radiation therapy. This review outlines the applicability of cetuximab and bevacizumab in the treatment of head and neck cancer as well as the clinical trials performed that give an idea about the efficacy and safety of these monoclonal antibodies. Based upon the literature reviewed, it can be deduced that immunotherapy is to be adopted and different targets are to be explored in it in order to combat head and neck cancer. Currently, immunotherapeutic drugs of two major targets have been discussed. These agents are eveneffective in combination with other therapeutic modalities that are not being able to achieve desirable outcomes due to issues such as resistance and toxicities. Thus, newer targets as well as newer agents acting on established targets are to be explored in order to improve disease outcomes.

Long-term survival in elderly women receiving chemotherapy for non-metastatic breast cancer: a population-based analysis.

Older women are poorly represented in trials evaluating chemotherapy for breast cancer (BC). This study aimed to describe survival and associated factors among elderly women receiving chemotherapy for non-metastatic BC. This was a population-based cohort study including women ≥ 70years old diagnosed with invasive, non-metastatic BC from 2010 to 2017 in SEER. Among those who received chemotherapy, overall survival (OS) was determined using Kaplan-Meier curves and hazard ratios were reported with 95% confidence intervals (CIs). Adjustment was made for available confounders. Co-morbidity is not available in SEER. BC-specific survival (BCSS) and subdistribution hazard ratios were determined using competing risks analysis. The cohort consisted of 109,239 women aged 70+, of whom 17,961 (16%) received chemotherapy. Chemotherapy patients were younger (median 73.0years vs.77.0), had more advanced disease (25% stage III vs.5.2%), and were more likely to receive mastectomy (50% vs.33%). Among chemotherapy patients, 5-year OS was 77.8% (95% CI 76.9-78.6%), and for women 80+ was 60.2% (95% CI 57.5-63.1%). More recent diagnoses, no previous history of cancer, and receipt of radiotherapy were all associated with improved BCSS. Conversely, older age, higher tumour grade, advanced stage, and human epidermal growth factors receptor (HER)2negativetumours were associated with worse BCSS. 56% of deaths were due to BC, and women aged 80+ had worse BCSS compared to those aged 70-79 (adjusted sdHR 1.62, 95% CI 1.43-1.84). Elderly women with advanced disease can achieve good survival after chemotherapy for non-metastatic BC. Those with HER2+ disease have superior survival, reinforcing benefit in this population.

A toolkit for recombinant production of seven human EGF family growth factors in active conformation

Epidermal growth factors (EGF) play a wide range of roles in embryogenesis, skin development, immune response homeostasis. They are involved in several pathologies as well, including several cancer types, psoriasis, chronic pain and chronic kidney disease. All members share the structural EGF domain, which is responsible for receptor interaction, thereby initiating transduction of signals. EGF growth factors have intense use in fundamental research and high potential for biotechnological applications. However, due to their structural organization with three disulfide bonds, recombinant production of these factors in prokaryotic systems is not straightforward. A significant fraction usually forms inclusion bodies. For the fraction remaining soluble, misfolding and incomplete disulfide bond formation may affect the amount of active factor in solution, which can compromise experimental conclusions and biotechnological applications. In this work, we describe a reliable procedure to produce seven human growth factors of the EGF family in Escherichia coli. Biophysical and stability analyses using limited proteolysis, light scattering, circular dichroism and nanoDSF show that the recombinant factors present folded and stable conformation. Cell proliferation and scratch healing assays confirmed that the recombinant factors are highly active at concentrations as low as 5 ng/ml.

Rule-Based Information Extraction from Free-Text Pathology Reports Reveals Trends in South African Female Breast Cancer Molecular Subtypes and Ki67 Expression

Clinical information on molecular subtypes and the Ki67 index is critical for breast cancer (BC) prognosis and personalised treatment plan. Extracting such information into structured data is essential for research, auditing, and cancer incidence reporting and underpins the potential for automated decision support. Herewith, we developed a rule-based natural language processing algorithm that retrieved and extracted important BC parameters from free-text pathology reports towards exploring molecular subtypes and Ki67-proliferation trends. We considered malignant BC pathology reports with different free-text narrative attributes from the South African National Health Laboratory Service. The reports were preprocessed and parsed through the algorithm. Parameters extracted by the algorithm were validated against manually extracted parameters. For all parameters extracted, we obtained accurate annotations of 83-100%, 93-100%, 91-100%, and 92-100% precision, recall, F1-score, and kappa, respectively. There was a significant trend in the proportion of each molecular subtype by patient age, histologic type, grade, Ki67, and race. The findings also showed significant association in the Ki67 trend with hormone receptors, human epidermal growth factors, age, grade, and race. Our approach bridges the gap between data availability and actionable knowledge and provides a framework that could be adapted and reused in other cancers and beyond cancer studies. Information extracted from these reports showed interesting trends that may be exploited for BC screening and treatment resources in South Africa. Finally, this study strongly encourages the implementation of a synoptic style pathology report in South Africa.

Recombinant Human Epidermal Growth Factor/Quatsome Nanoconjugates: A Robust Topical Delivery System for Complex Wound Healing (Adv. Therap. 6/2021)

In article number 2000260, Jaume Veciana, Nora Ventosa and co-workers report a soft, reliable, and scalable method based on compressed CO2 for obtaining nanoconjugates of recombinant human epidermal growth factors and nanovesicles. These nanoconjugates exhibit appropriate physicochemical properties together with an antimicrobial activity preventing infections that promote complete closure of complex wounds. Therefore, such nanoconjugates are a potential nanomedicine for the topical treatment of complex wounds, particularly diabetic foot ulcers.

Modeling Precision Cardio-Oncology: Using Human-Induced Pluripotent Stem Cells for Risk Stratification and Prevention.

Purpose of ReviewCardiovascular toxicity is a leading cause of mortality among cancer survivors and has become increasingly prevalent due to improved cancer survival rates. In this review, we synthesize evidence illustrating how common cancer therapeutic agents, such as anthracyclines, human epidermal growth factors receptors (HER2) monoclonal antibodies, and tyrosine kinase inhibitors (TKIs), have been evaluated in cardiomyocytes (CMs) derived from human-induced pluripotent stem cells (hiPSCs) to understand the underlying mechanisms of cardiovascular toxicity. We place this in the context of precision cardio-oncology, an emerging concept for personalizing the prevention and management of cardiovascular toxicities from cancer therapies, accounting for each individual patient’s unique factors. We outline steps that will need to be addressed by multidisciplinary teams of cardiologists and oncologists in partnership with regulators to implement future applications of hiPSCs in precision cardio-oncology.Recent FindingsCurrent prevention of cardiovascular toxicity involves routine screenings and management of modifiable risk factors for cancer patients, as well as the initiation of cardioprotective medications. Despite recent advancements in precision cardio-oncology, knowledge gaps remain and limit our ability to appropriately predict with precision which patients will develop cardiovascular toxicity. Investigations using patient-specific CMs facilitate pharmacological discovery, mechanistic toxicity studies, and the identification of cardioprotective pathways. Studies with hiPSCs demonstrate that patients with comorbidities have more frequent adverse responses, compared to their counterparts without cardiac disease. Further studies utilizing hiPSC modeling should be considered, to evaluate the impact and mitigation of known cardiovascular risk factors, including blood pressure, body mass index (BMI), smoking status, diabetes, and physical activity in their role in cardiovascular toxicity after cancer therapy. Future real-world applications will depend on understanding the current use of hiPSC modeling in order for oncologists and cardiologists together to inform their potential to improve our clinical collaborative practice in cardio-oncology.SummaryWhen applying such in vitro characterization, it is hypothesized that a safety score can be assigned to each individual to determine who has a greater probability of developing cardiovascular toxicity. Using hiPSCs to create personalized models and ultimately evaluate the cardiovascular toxicity of individuals’ treatments may one day lead to more patient-specific treatment plans in precision cardio-oncology while reducing cardiovascular disease (CVD) morbidity and mortality.

EGFR and HER2 Expression in Primary Ovarian High-Grade Serous Carcinoma and Their Prognostic Value

Background: Human epidermal growth factors receptors such as EGFR and HER2 play an important role in tumorigenesis and are used as therapeutic targets. Their role in aggressive primary ovarian high-grade serous carcinoma (HGSC) is controversial. Aim: To study the expression of EGFR and HER2 in ovarian HGSC, to correlate their expression with other clinicopathological parameters and to study their prognostic value. Methods: Imunohistochemical staining of EGFR, HER2 and Ki-67 was done for 54 ovarian HGSC specimens. According to the used scoring methods, the expression of EGFR and HER2 was classified as high or low. Results: High expression of EGFR and HER2 was found in a minority of specimens; 39% and 15%, respectively. None of the studied clinicopathological parameters correlated significantly with the expression of EGFR and HER2, except for the carcinoembryonic antigen level which correlated positively with HER2 expression. Disease-free survival of patients did not differ significantly according to the level of expression of EGFR and HER2 (p =0.684 and 0.186, respectively). Similarly, overall survival did not differ significantly (p =0.911 and 0.346, respectively). Conclusion: The results of this study suggest that the prognostic value of EGFR and HER2 in ovarian HGSC is questionable.

Enhancement of Skin Wound Healing by rhEGF-Loaded Carboxymethyl Chitosan Nanoparticles.

The self-assembly of hydrophobically modified polymers has become a research hotspot due to its wide application in the biomedical field. Recombinant human epidermal growth factors (rhEGFs) are molecules that are able to enhance wound healing; however, they have a short half-life and require sustained action to enhance their mitogenic effect on epithelial cells. Here, we proposed a new delivery system to avoid the inhibition of rhEGF by various enzymes, thus improving its bioavailability and sustained release. The amphiphilic polymer was composed of conjugated linoleic acid (CLA) and carboxymethyl chitosan (CMCS), which were further characterized by fourier transformed infrared spectroscopy (FTIR) and 1H nuclear magnetic resonance (1H NMR). Then, the self-assembly behavior of CLA–CMCS (CC) polymer in water was observed in which the particle size of CC decreased from 196 to 155 nm with the degree of CLA substitution increasing. The nanoparticles were loaded with rhEGF and the maximum rhEGF loading efficiency (LE) of CC3 nanoparticles was 82.43 ± 3.14%. Furthermore, CC nanoparticles (NPs) exhibited no cytotoxicity for L929 cells, and cell proliferation activity was well preserved after rhEGF loading to CC-NPs and was comparable to that of free rhEGF. Topically applied rhEGF:CC-NPs significantly accelerated the wound-closure rate in full thickness, which was most probably due to its sustained release and enhanced skin permeation. In conclusion, carboxymethyl chitosan-based nanoparticles were constructed and showed good cytocompatibility. Moreover, these findings also demonstrated the therapeutic potential of rhEGF:CC-NPs as a topical wound-healing drug carrier.

Expression of HER2 and EGFR Proteins in Advanced Stage High-grade Serous Ovarian Tumors Show Mutual Exclusivity.

Human epidermal growth factors play an important role in ovarian carcinogenesis and are evaluated for prognostic and possible therapeutic roles in high-grade serous ovarian malignancies. The present study was undertaken to evaluate the expression of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) in advanced stage serous carcinoma and their influence on prognosis. The expression of HER2 and EGFR was studied in 59 cases of stage III and IV ovarian serous carcinomas by immunohistochemistry and fluorescent in situ hybridization. Of the 48 interpretable tumors for HER2, 6 tumors (12.5%) were scored as positive, 14 (29%) as equivocal and 28 tumors (58.5%) were negative by immunohistochemistry, while only 2/48 (4%) showed frank amplification by fluorescent in situ hybridization with ≥4 copies per cell. HER2 gene expression measured by quantitative polymerase chain reaction had good positive correlation with both protein expression and gene amplification. Although EGFR expression was seen in 32% of tumors, none of the tumors positive for HER2 protein or gene amplification had co-expression of EGFR indicating mutual exclusivity of their expression. Gene expression of both proteins also confirmed their inverse correlation (Pearsons CC=-0.15, P=0.3). Further there was no influence of protein or gene expression of these markers on the overall survival. In conclusion, HER2 and EGFR are expressed in a small percentage of tumors and the mutual exclusivity of these markers precludes the possibility of dual targeting with anti-HER2 and anti-EGFR therapy in advanced stage high-grade serous ovarian carcinoma.

Breast Cancer: A Molecularly Heterogenous Disease Needing Subtype-Specific Treatments.

Breast cancer is the most commonly occurring cancer in women. There were over two-million new cases in world in 2018. It is the second leading cause of death from cancer in western countries. At the molecular level, breast cancer is a heterogeneous disease, which is characterized by high genomic instability evidenced by somatic gene mutations, copy number alterations, and chromosome structural rearrangements. The genomic instability is caused by defects in DNA damage repair, transcription, DNA replication, telomere maintenance and mitotic chromosome segregation. According to molecular features, breast cancers are subdivided in subtypes, according to activation of hormone receptors (estrogen receptor and progesterone receptor), of human epidermal growth factors receptor 2 (HER2), and or BRCA mutations. In-depth analyses of the molecular features of primary and metastatic breast cancer have shown the great heterogeneity of genetic alterations and their clonal evolution during disease development. These studies have contributed to identify a repertoire of numerous disease-causing genes that are altered through different mutational processes. While early-stage breast cancer is a curable disease in about 70% of patients, advanced breast cancer is largely incurable. However, molecular studies have contributed to develop new therapeutic approaches targeting HER2, CDK4/6, PI3K, or involving poly(ADP-ribose) polymerase inhibitors for BRCA mutation carriers and immunotherapy.

Abstract B47: Associations between clinicopathological features and breast cancer subtypes

Abstract Background: Breast cancer (BC) exists as several heterogeneous subtypes with differing distributions, risk factors, tumor behaviors and clinical outcomes. The risk factors that are associated with and the clinicopathological features distinguishing BC subtypes remain undefined. In this study, we examined associations between BC risk factors and tumor clinicopathological features, and molecular subtypes of BC. Methods: A cohort of 629 invasive BC cases from New Jersey (51.2% white; 48.8% African American [AA]) were selected from the Breast Cancer Treatment Study, who originally participated in the Women's Circle of Health Study. These women were diagnosed with stage I, II, or T3N1M0 BC between 2005 and 2010. In the present study, we examined BC risk factors (from interview-administered questionnaire data) and tumor clinicopathological features (from abstraction of detailed medical records), and their associations with BC molecular subtypes (classified by estrogen receptor [ER], progesterone receptor [PR] and human epidermal growth factors receptor 2 [HER2] expression patterns as defined in pathology records). BCs were classified into four mutually exclusive subtypes: ER+/PR+/HER2- (luminal A), ER+/PR+/HER2+, ER-/PR-/HER2+ (non-luminal HER2 expressing), and ER-/PR-/HER2- (triple negative [TN]). Multinomial logistic regression models (with luminal A as the reference group) were used to examine the associations between tumor features and BC subtype. Results: In this study, the largest proportion of BC cases was diagnosed with luminal A (68.4%), followed by TN (14.5%), ER+/PR+/HER2+ (11.1%), and non-luminal HER2 expressing (6.0%) tumors. Younger age (<45 years) was associated with increased odds of ER+/PR+/HER2+ (Odds Ratio [OR] 2.1, 95% CI: 1.2-3.8) and TN (OR 1.7, 95% CI: 1.0-2.9) tumors. AAs were more likely to have ER+/PR+/HER2+ (OR 1.9, 95% CI: 1.1-3.1) and TN (OR 2.7, 95% CI: 1.7-4.3) tumors. Lower level of education (less than college vs. college graduate or higher: OR 1.6, 95% CI: 1.0-2.7) and lower income ($70,000 vs. ≥$70,000: OR 1.8, 95% CI: 1.1-3.2) were associated with increased odds of TN tumors. Compared to the luminal A subtype, there were increased odds of self-detection (as the mode of initial tumor detection) among ER+/PR+/HER2+ (OR 1.8, 95% CI: 1.1-3.1), non-luminal HER2 expressing (OR 2.4, 95% CI: 1.2-4.7), and TN (OR 2.7, 95% CI: 1.7-4.2) tumors. These three subtypes were also associated with poorer differentiation (ER+/PR+/HER2+, OR 3.3, 95% CI: 1.9, 5.6; non-luminal HER2 expressing, OR 16.7, 95% CI: 6.7-41.5; and TN, OR 13.8, 95% CI: 7.9-24.4); higher AJCC stage (ER+/PR+/HER2+, OR 1.9, 95% CI: 1.1, 3.2; non-luminal HER2 expressing, OR 2.6, 95% CI: 1.3-5.2; and TN, OR 1.8, 95% CI: 1.2-2.9); larger tumor size (>2.0 cm vs. ≤1.0 cm: ER+/PR+/HER2+, OR 2.5, 95% CI: 1.3-4.7; non-luminal HER2 expressing, OR 3.7, 95% CI: 1.6-8.7; and TN, OR 3.9, 95% CI: 2.1-7.5); p53 expression (ER+/PR+/HER2+, OR 2.1, 95% CI: 0.7-6.1; non-luminal HER2 expressing, OR 3.3, 95% CI: 1.0-10.4; and TN, OR 5.0, 95% CI: 2.3-10.8); and Ki67 expression (ER+/PR+/HER2+, OR 2.6, 95% CI: 1.5-4.5; non-luminal HER2 expressing, OR 2.7, 95% CI: 1.3-5.5; and TN, OR 4.2, 95% CI: 2.6-6.7) as compared to luminal A tumors. Conclusion: These findings support an association between sociodemographics and BC subtypes, specifically showing increased odds of TN tumors among women who are younger, AA, and/or of lower socioeconomic status. These findings also suggest that the BC subtypes that are commonly observed among racial/ethnic minorities and economically disadvantaged groups exhibit more aggressive clinicopathological features and are associated with an increased likelihood of self-detection. Citation Format: Adana A.M. Llanos, Sheenu Chandwani, Kim M. Hirshfield, Yong Lin, Elisa V. Bandera, Christine B. Ambrosone, Kitaw Demissie. Associations between clinicopathological features and breast cancer subtypes. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B47.

Topically applied human like epidermal growth factors in aging skin: Randomized, placebo controlled trail by evaluating skin physiology and skin surface parameters

Topically applied human like epidermal growth factors in aging skin: Randomized, placebo controlled trail by evaluating skin physiology and skin surface parameters

High serum HER2 extracellular domain levels: correlation with a worse disease-free survival and overall survival in primary operable breast cancer patients

High serum human epidermal growth factors receptor-2 (HER2) extracellular domain (ECD) has been identified as an independent prognostic indicator of poor prognosis in metastatic breast cancer. However, its prognostic value in primary operable breast cancer was still controversial. We aim to investigate the correlation between serum HER2 ECD levels and tissue HER2 status, the association between serum HER2 ECD levels and clinicopathological characteristics, and their impacts on disease-free survival (DFS) and overall survival (OS) in primary operable breast cancer. Two hundred and fifty-two primary operable breast cancer patients pretreated from 2002 to 2009 in Sun Yat-Sen University Cancer Center were enrolled in this study. Serum HER2 ECD was measured by chemiluminescent assay, and tissue HER2 status was accessed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) assay. There was a significant correlation between serum HER2 ECD levels and HER2 tissue status (P < 0.001, R = 0.36). High serum HER2 ECD levels (≥15 ng/mL) were significantly associated with age (≥35 years) (P = 0.028), postmenopausal status (P < 0.001), stage III (P < 0.001), tumor size (≥2 cm) (P < 0.001), lymph node involvement (P < 0.001), negative estrogen receptor (P = 0.005), and progesterone receptor status (P = 0.001). Multivariate analysis showed that high serum HER2 ECD level was an independent prognostic factor of worse DFS (P = 0.014) and OS (P = 0.014) in primary operable breast cancer patients. Serum HER2 ECD level can reflect tissue HER2 status and can be an independent prognostic indicator for primary operable breast cancer patients.

An epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea

Three peptide toxins (gigantoxins I–III) with crab toxicity were isolated from the sea anemone Stichodactyla gigantea by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T and their complete amino acid sequences were determined. Gigantoxins II (44 residues) and III (48 residues) have LD 50 (against crabs) of 70 and 120 μg/kg, respectively, and are analogous to the known type 1 and 2 sea anemone sodium channel toxins, respectively. On the other hand, gigantoxin I (48 residues) is potently paralytic to crabs (ED 50 215 μg/kg), although its lethality is very weak (LD 50>1000 μg/kg). Interestingly, gigantoxin I has 31–33% homologies with mammalian epidermal growth factors (EGFs), with the same location of six cysteine residues. In accordance with the sequence similarity, gigantoxin I exhibits EGF activity as evidenced by rounding of A431 cells and tyrosine phosphorylation of the EGF receptor in the cells, although much less potently than human EGF. Gigantoxin I is the first example of EGF-like toxins of natural origin.

Cytoplasmic competence of reconstructed GV-oocytes can be achieved with in-vitro maturation (IVM).

Objective: Fertilization and embryo development potential of reconstructed oocytes after GV transfer may be greatly compromised by a lack of critical cytoplasmic factors. Human chorionic gonadotropins (hCG) and epidermal growth factors (EGF) have been demonstrated to induce final maturation of preantral follicles in-vitro. The aim of this investigation was to assess whether grafted GV-oocytes can be matured in-vitro to gain full competence for oocyte maturation, fertilization and embryogenesis.

Cell Growth Activity of Growth-Blocking Peptide

Growth-blocking peptide (GBP) is an insect biogenic peptide that retards the development of lepidopteran larvae. cDNAs encoding GBP were isolated from three lepidopteran species:Pseudaletia separata, Mamestra brassicaeandSpodoptera litura.Comparison of these molecules revealed that the GBP coding regions were 70% homologous. In contrast, the upstream regions of the deduced propeptides were only 33 % identical. Sequence analysis further suggested that GBP shares some structural similarities with human epidermal growth factors. Bioassay data revealed that several pmol/ml of GBP stimulated DNA synthesis of a human keratinocyte cell line and of SF-9 insect cells. However, several nmol/ml of GBP did not stimulate cell proliferation at all.In vivostudies similarly indicated that low concentrations of GBP stimulated larval growth while high concentrations of GBP retarded growth. These data suggest that GBP acts as a growth factor that regulates insect larval development.

Development and validation of two solid-phase enzyme immunoassays (ELISA) for quantitation of human epidermal growth factors (hEGFs).

The purpose of the present investigation was to develop and validate two separate enzyme-linked immunosorbent assays (ELISA) for quantitation of exogenous human epidermal growth factor (hEGF1-53) and its truncated fragment (hEGF1-48) in rat plasma. The present assay systems were based on the sandwiching of the antigen between a monoclonal mouse anti-hEGF1-53 antibody, pre-coated on a 96-well polystyrene plate, and a polyclonal rabbit anti-hEGF1-48 antibody, which is then detected with a peroxidase-labeled goat anti-rabbit antibody. The calibration curves for hEGF1-48 and hEGF1-53 in plasma were validated over a concentration range of 7.8-250 and 62.5-1000 pg/ml, respectively. Determined from replicate assays of hEGF1-48 quality control samples, the intra-assay precision and accuracy were < or = 8.8% RSD and within +/- 9.8%; and the inter-assay precision and accuracy were < or = 14.8% RSD and within +/- 9.7% RE, respectively. Determined from replicate assays of hEGF1-53 quality control samples, the intra-assay precision and accuracy were < or = 10.0% RSD and within +/- 8.5%; and the inter-assay precision and accuracy were < or = 10.0% RSD and within +/- 5.7% RE, respectively. The limit of quantitation of the hEGF1-48 and hEGF1-53 assay using 200 microliters plasma per well is 7.8 and 62.5 pg/ml, respectively. These two ELISA methods are specific to hEGFs and do not cross-react with mouse EGF or other growth factors (TGF alpha, TGF beta, PDGF, and FGF) or lymphokines (IL1 beta and TNF alpha). These validated methods have been routinely applied to assay of plasma samples from various pharmacokinetic studies in rats receiving intravenous hEGFs. Both assay methods were also adapted to assay endogenous hEGFs in biological fluids of different animal species. Two sensitive ELISA methods have been validated for quantitation of hEGF1-53 and hEGF1-48 in rat plasma. Their utility has been demonstrated in the application of assaying immunoreactive concentration of exogenous and endogenous epidermal growth factors.

Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma

A sensitive assay was developed for human epidermal growth factors (hEGF) 1–48 (dosed), hEGF 1–53 (endogenous), without interference from potential metabolites hEGFs 1–47 or 1–46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 μg ml −1, although a detection limit of 75 pg ml −1 appears feasible.

Structural characterization and biological activity of recombinant human epidermal growth factor proteins with different N-terminal sequences

The primary structures and molecular homogeneity of recombinant human epidermal growth factors from different suppliers were characterized and their biological activities evaluated by a standard DNA synthesis assay. Molecular weight determinations using 252Cf-plasma-desorption and electrospray mass spectrometry in combination with N- and C-terminal sequence analysis and determination of intramolecular disulfide bridges revealed that one recombinant protein had the correct human-identical structure (54 aa residues; 6347 Da). In contrast, a second recombinant protein (7020 Da) was found to contain a pentapeptide (KKYPR) insert following its N-terminal methionine. This structural variant showed a significant reduction in its capacity to stimulate DNA synthesis.