Growth Factor Binding Protein Research Articles

Proteomic analysis of human amniotic fluid from Rh− pregnancy

The objective of this study was to identify proteins from the amniotic fluid of the most common reproductive ailment, that is, Rh(-) incompatibility. In the present study, AF from Rh(-) pregnancy in the 29-35th week of gestation was analyzed by 2-DE, and our purpose was to identify the major spots on 2D gel. Proteins from an amniotic fluid sample was separated on 2D gel and visualized after silver staining. Most of the spots were analyzed by MALDI-TOF/MS, and were identified based on the NCBI database by a peptide mass fingerprinting method. Albumin, serotransferrin, haptoglobin, alpha-fetoprotein and immunoglobulin were the major proteins detected. On comparison with 2D gel of normal AF, differences were observed at the level of albumin, and newly identified Calgranulins and Neutophil-defensins were observed. Many enzymes, growth factor binding proteins, protease inhibitors and proteins related to inflammation have also been identified. This study demonstrates, for the first time, the potential utility of proteomics-based approaches to identify specific biomarkers like Calgranulins and Neutophil-defensins and diagnostic profiles for Rh(-) incompatibility processes and pathophysiologic conditions of pregnancy.

Insulin like growth factor‐1 and insulin like growth factor binding proteins in the cerebrospinal fluid and serum from patients with Alzheimer's disease

Insulin like growth factor-1 (IGF-1) is ubiquitously expressed growth factor that has profound effects on the growth and differentiation of many cell types and tissues, including cells of the central nervous system (CNS). IGF-1 is produced by a wide variety of cells and is found in many biological fluids including cerebrospinal fluid (CSF). IGF-1 plays important role during CNS development and repair. IGF-1 has broad range neuroprotective effects and is a therapeutic candidate for Huntington's disease (HD). IGF-1 protects striatal neurons from the toxicity of mutated Huntington in vitro and improves neuronal survival in vivo in a phenotypic model of HD. Alzheimer's disease (AD) is an age-dependent dementia characterized by progressive loss of cognitive functions and by characteristic pathological changes in the brain: the formation of aggregates extracellularly by beta-amyloid (AB) peptide and intracellularly by tau proteins. Since cerebrospinal fluid (CSF) is in contact with the extracellular space of the brain, biochemical brain modifications could be reflected in the CSF. IGFs in circulation and other physiological fluids are associated with a group of high-affinity binding proteins insulin like growth factor binding proteins (IGFBPs) that specifically bind and modulate their bioactivity at the cellular level. The aim of this study was to determine the level of CSF and serum IGF-1 and IGFBPs concentrations in the patients with AD. CSF was obtained by lumbar puncture. The presence of IGF-1 and IGFBPs in the CSF and serum samples was confirmed by Western blot using anti-IGF-1 and IGFBPs antibodies. Using enzyme linked immunosorbent assay (ELISA), it was shown that the concentration of CSF and serum IGF-1 and IGFBPs in the patients with AD is higher than in normal control. The data from this study indicate that IGF-1 is a constant component of human CSF. It is also concluded that high levels of CSF IGF-1 may be partly related to AD pathophysiology.

The relationship between adiponectin, leptin, insulin, insulin-like growth factor and IGF binding protein-3 in cord blood and neonatal anthropometric parameters

Purpose : This study was designed to examine the effects of adiponectin, leptin, insulin, insulin-like growth factor (IGF)-I and IGF binding protein (BP)-3 levels in cord blood on weight, length, and adiposity at birth in healthy term infants. In addition, we evaluated the mechanism to change the hormone levels in appropriate for gestational age (AGA) during the first month. Methods : We collected cord blood from 200 term neonates (109 males, 91 females) with no perinatal problems, and measured the hormone levels and anthropometric parameters including weight, length, and skin-fold thickness. Term neonates were divided into 3 groups as follows: birth weight appropriate for gestational age (AGA) (n=132), birth weight less for gestational age (SGA) (n=29), and birth weight more for gestational age (LGA) (n=39). Venous blood samples of 15 fullterm healthy neonates were obtained at 3, 7, and 30 d after birth. Results : The adiponectin, insulin, and IGF-I levels were significantly lower in the SGA group than in the AGA and LGA groups. The leptin levels were significantly higher in the LGA group than in the AGA and SGA groups. Cord blood adiponectin, leptin, insulin, IGF-I, and IGFBP-3 levels correlated significantly and positively with birth weight and the sum of the skin-fold thickness. A significant positive correlation was observed between adiponectin, leptin, and IGF-I levels and birth weight. Adiponectin level correlated significantly with that leptin level (r=0.191, P=0.038), but not with insulin, IGF-I and IGFBP-3 levels. IGF-I levels were higher in females than in males. At 7 d after birth, the leptin level decreased along with physiologic weight loss, and then increased. IGF-I, also decreased at 3 d, significantly increased 1 month later. Conclusion : We suggest that adiponectin, leptin, insulin, IGF-I, and IGFBP-3 play an important role in regulating fetal growth. Adiponectin may be involved in regulating fetal growth through mechanisms different from those mediated by insulin or IGF-I. High levels of IGF-I in female neonates indicates a gender difference which serves as evidence for in utero sexual dimorphism. It is likely that IGF-I has a more important role than that of hormones in postnatal growth.

Insulin like growth factor binding proteins - evidence towards a tumour suppressive function in human breast cancer

Insulin like growth factor binding proteins - evidence towards a tumour suppressive function in human breast cancer

Mechanistic insights into the inhibition of prostate specific antigen by β‐lactam class compounds

Prostate Specific Antigen (PSA) is a biomarker used in the diagnosis of prostate cancer and to monitor therapeutic response. However, its precise role in prostate carcinogenesis and metastasis remains largely unknown. A number of studies arguing in the favor of an active role of PSA in prostate cancer development and progression have implicated this serine protease in the release and activation of growth factors such as insulin-like growth factor 1 (IGF1) through cleavage of insulin like growth factor binding protein 3 and Transforming Growth Factor beta (TGF-beta) through cleavage of Latent TGF-beta. In contrast, other studies suggest that PSA activity might hinder tumor development and progression. In light of these contradictory findings, efficient inhibitors of PSA are needed for exploring its biological role in tumor development and metastasis. Towards the goal of developing potent inhibitors of PSA, we have explored the molecular mechanism of a series of beta-lactam based compounds on binding to PSA using activity assays, matrix assisted laser desorption ionization with a time-of-flight mass spectrometry, and GOLD docking methodology. The mass spectrometry experiments and the activity assays confirmed the time-dependent and covalent nature of beta-lactam binding. To gain insights on the reaction intermediates at the molecular level, we docked beta-lactam inhibitors to a homology modeled PSA using the GOLD docking program in noncovalent and covalent binding modes. The docking studies elucidated the molecular details of the early noncovalent Michaelis complex, the acyl-enzyme covalent complex, and the nature of conformational reorganization required for the long term stability of the covalent complex. Additionally, the molecular basis for the effect of stereochemistry of the lactam ring on the inhibitory potency was elucidated through docking of beta-lactam enantiomers. As a validation of our docking methodology, two novel enantiomers were synthesized and evaluated for their inhibitory potency using fluorogenic substrate based activity assays. Additionally, cis enantiomers of eight beta-lactam compounds reported in a previous study were docked and their GOLD scores and binding modes were analyzed in order to assess the general applicability of our docking results. The close agreement of our docking results with the experimental data validates the mechanistic insights revealed through the docking studies and paves the way for the design and development of potent and specific inhibitors of PSA.

Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes

Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfbeta and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfbeta ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfbeta receptors, consistent with paracrine Tgfbeta signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.

Differential expression of insulin like growth factor binding protein-4 (IGFBP4) in melanoma progression

8557 Background: Members of the IGFBP family are thought to regulate tumor progression through both IGF-dependent and independent mechanisms. Moreover, IGFBPs may exhibit distinct functions depending on whether they are present locally in tissue or systemically. Our preclinical data suggest that IGFBP4 serves as an endogenous inhibitor of angiogenesis and melanoma tumor growth. To examine the clinical validity of our observations, we compared IGFBP4 expression in tissue and sera in primary melanoma patients with a low risk of metastases versus patients with established metastatic disease. Methods: Eighty-nine melanoma patients (primary stage IA, n=57; metastatic, n=32; 44 M, 45 F; Median Age=56) prospectively enrolled in the Interdisciplinary Melanoma Cooperative Group at the NYU School of Medicine were studied. IGFBP4 protein expression in tumors was assessed in 83 patients using goat anti-human IGFBP4 antibody (R&D Systems) in an immunohistochemical assay. For each case, the percentage of tumor cells that stained positively was estimated. IGFBP4 concentration in the serum was measured in 64 patients by an ELISA assay (Diagnostic Systems Laboratories, Inc.). Fifty-eight patients had both sera and tumor specimens available for correlation. Results: The median IGFBP4 expression in tumor specimens from primary patients was 70% compared to 5% in metastatic patients (P=0.002, Wilcoxon rank-sum test). The median concentration of IGFBP4 in sera was 43.8 ng/ul in primary patients versus 41.0 ng/ul in metastatic patients (P=0.83). A weak association between IGFBP4 expression in tissue and IGFBP4 shedding in sera was observed (Spearman correlation coefficient=0.16, P=0.24) in the 58 cases with both sera and tissue available. Conclusions: Our data demonstrate that IGFBP4 expression in melanoma clinical specimens decreases in the progression from primary to metastatic melanoma, consistent with its potential role as an inhibitor of angiogenesis. Data also suggest that IGFBP4 tissue expression may be more clinically relevant than its sera shedding. Future studies using a larger sample size and extended follow up are needed to further define the role of IGFBP4 in melanoma progression. No significant financial relationships to disclose.

The use of gene array analysis to determine down-stream molecular expression patterns of trastuzumab (T) treatment

14135 Background: Breast cancers expressing the HER/2 amplicon are more aggressive and are associated with a poorer prognosis. HER/2 over-expression has been correlated with expression of proteins responsible for invasion, metastases, rapid growth, angiogenesis and apoptosis. However, it is unknown if T treatment of HER/2 over-expressing tumors reverses these phenotypes. Methods: BT 474 and SK-BR3 breast cancer cell lines contain the HER/2 amplicon. Under growth conditions demonstrating inhibitory effects of T by MTT and 3H-Thymidine incorporation, (0.25–50 micrograms/ml for up to 9 days), RNA extracts from treated and un- treated cells were analyzed and compared by whole human genome, gene array analysis. Results: Specific gene products reported to be up-regulated in HER/2 over-expressed breast cancer lines or responsible for aggressive behavior were assessed for down regulation by T treatment including: topoisomerase II, heregulin, egfr-1,2,3 and 4, p27, ki67, mapKinase, akt, caspase-3, caspase-7, caspase-9, bcl-2, veg-f, veg-f receptor, p38, sapk/jnk, erk1/2, LGALS1 galectin-1, LGALS3 galectin-3, proline-4-hydrolase P4HA2, FN1 fibronectin -1, CDH3 p-cadherin, laminin receptor protein -1, pro2605 and insulin like growth factor binding protein-3. No significant change in these mRNA levels were observed due to T treatment. In contrast, over 1000 other gene were found to be specifically and significantly up or down regulated including the down regulation of S100 Calcium binding protein associated with increase metastatic potential. Conclusions T treatment does not reverse the expression of a large array of proteins conferring poor prognostic features on HER/2+ tumor. The use of gene array analysis to study T induced gene expression is a robust method to assess downstream protein expression patterns mediated by antibody binding to receptor. No significant financial relationships to disclose.

Phase II prevention trial of celecoxib in women at increased risk for breast cancer

1501 Background: Tamoxifen and raloxifen reduce the risk of estrogen receptor (ER) positive breast cancer. Acceptance of SERMs is low due to toxicities. New agents with a better toxicity profile which also will prevent ER-negative breast cancer are needed. The cyclooxygenase II inhibitor celecoxib has antiproliferative, and antiangiogenetic properties and is a putative breast cancer prevention agent. Our objective in this prospective short-term prevention study was to evaluate the effect of celecoxib on biomarkers in breast tissue and serum of high risk women. Methods: High risk individuals with a history of atypical hyperplasia, or lobular carcinoma insitu, or Gail risk greater than 1.67%, or previous history of ER negative breast cancer were eligible. After signing informed consent subjects underwent baseline fine needle aspiration (FNA) of the breast and serum collection for biomarker analysis. Subjects started celecoxib at 400 mg po BID for 6 months and underwent repeat FNA and serum collection. Biomarker endpoints included changes in ER and KI-67 expression in breast tissue analyzed by immunohistochemistry and insulin like growth factor binding protein (IGFBP-1) in serum analyzed by ELISA. The difference in biomarkers before and after treatment was assessed using a Wilcoxon signed rank test. Results: 49 patients were enrolled and accrual has been completed. Median age was 51 years and 57% of women were postmenopausal. The mean pre- and post treatment ER expression was 35.5% and 31%, respectively. The difference in ER expression was not significant (p = 0.1). The mean pre- and post treatment KI-67 expression was 1.68% and 1.60%, respectively. The difference was not significant (p = 0.8). However, there was a statistically significant difference in pre- and post treatment IGFBP-1 levels (p=0.023), with pre-and post treatment levels being 6.81ng/ml and 11.51ng/ml, respectively. No grade 3 or 4 toxicities were observed and there was no drop-out from the study. Conclusions: We have found a significant modulation of IGFBP-1 levels with 6 months celecoxib treatment in women who are at increased risk of developing breast cancer. The acceptable side effect profile and modulation of the biomarker (IGFBP-1) provides support for continued evaluation of celecoxib as a breast cancer prevention agent. No significant financial relationships to disclose.

Burn Injury Reveals Altered Phenotype in Mannan-Binding Lectin-Deficient Mice

Burn injury destroys skin, the second largest innate immune organ in the body, and triggers chaotic immune and inflammatory responses. The pattern recognition molecule, mannan-binding lectin (MBL), plays an important role in the first-line host defense against infectious agents. MBL initiates the lectin complement pathway and acts as an opsonin. Recent studies suggest that MBL also modulates inflammatory responses. We report that local responses after burn in MBL null mice differ from those found in wild-type (WT) mice in the following important biological markers: spontaneous eschar separation, thinned epidermis and dermis, upregulation of soluble factors including cytokines, chemokines, cell adhesion molecules, a growth factor-binding protein, and matrix metalloproteinases. Mice lacking C1q, C4, or C3 did not show the lack of eschar separation seen in MBL null-burn phenotype. These findings implicate MBL as an important molecule in the maintenance of the homeostatic balance.

Silencing of Insulin-like Growth Factor-binding Protein-2 in Human Glioblastoma Cells Reduces Both Invasiveness and Expression of Progression-associated Gene CD24

Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by rapid growth and extensive invasiveness. Overexpression of insulin-like growth factor-binding protein-2 (IGFBP-2) has been reported in GBM. However, it remains to be determined how IGFBP-2 is involved in the progression of GBM. We utilized short hairpin-RNA (shRNA) expression retroviral vectors to inactivate the IGFBP-2 gene permanently in two human GBM cell lines, U251 and YKG-1. The stable knockdown of IGFBP-2 resulted in decreased invasiveness, decreased saturation density of the cells in vitro, and decreased tumorigenicity in nude mice. Transcriptional profiling of both lines revealed several genes that were significantly down-regulated by inactivation of IGFBP-2. One such gene was CD24, which has been implicated in progression of various cancers. Indeed, CD24 was expressed in most GBM cases and the inactivation of CD24 in GBM cells suppressed cellular invasiveness, as was the case for IGFBP-2. Forced overexpression of CD24 led to increased invasiveness of both IGFBP-2-inactivated GBM cell lines and also A172, a human GBM cell line with low endogenous CD24. Further supporting the inter-relationship between IGFBP-2 and CD24, knockdown of IGFBP-2 suppressed the CD24 promoter activity. Moreover, both CD24 promoter activity and in vitro invasiveness were restored in knockdown cells by transfection with an IGFBP-2 expression plasmid. These results indicate that CD24 is modulated by IGFBP-2 and contributes to IGFBP-2-enhanced invasiveness of GBM cells.

Insight into redox-regulated gene networks in vascular cells.

To understand the complex nature of the atherogenic response initiated by oxidative stress in vascular smooth muscle cells (vSMCs), computational prediction methodology was employed to define putative gene-gene and gene-environment interactions in vSMCs subjected to oxidative chemical stress. Computational relationships were derived from the global gene expression profiles of murine cells challenged with a chemical pro-oxidant to cause oxidative stress or cells treated with anti-oxidant prior to oxidative injury. Target clones were chosen based on their biological relevance within the context of the atherogenic response and included lysyl oxidase, matrix metalloproteinase 2, insulin like growth factor binding protein 5, and lymphocyte antigen 6c. Established biological relationships were derived computationally confirming the usefulness of the algorithm in uncovering novel biological relationships worthy of future investigation. Thus, the predictive algorithm can be a useful tool to advance the frontiers of biological discovery.

Matrix metalloproteinase‐7 triggers the matricrine action of insulin‐like growth factor‐II via proteinase activity on insulin‐like growth factor binding protein 2 in the extracellular matrix

Many growth factors and cytokines are immobilized on the extracellular matrix (ECM) by binding to glycosaminoglycans and are stored in an inactive form in the cellular microenvironment. However, the mechanisms of ECM-bound growth factor or cytokine activation have not been well documented. We showed that the insulin-like growth factor type-1 receptor (IGF-1R) was rapidly phosphorylated after the addition of matrix metalloproteinase (MMP)-7 to a serum-starved human colon cancer cell line (HT29) and that phosphorylation was completely inhibited by an IGF-II neutralizing antibody. In the ECM of this cell line, IGF-II and IGF binding protein (BP)-2 coexisted, but IGFBP-2 disappeared from the ECM fraction after treatment with MMP-7 or heparinase III. On the other hand, in a cell line in which IGF-1R was overexpressed, IGF-1R was phosphorylated by supernatant from the MMP-7-treated ECM fraction of HT29 but not by that from a heparinase-III-treated ECM fraction. We also demonstrated that MMP-7 degrades IGFBP-2 in vitro at three cleavage sites (peptide bonds E(151)-L(152), G(175)-L(176) and K(181)-L(182)), which have not been documented previously. Taken together, these results demonstrate that MMP-7 generates bioactive IGF-II by degrading the IGF-II/IGFBP-2 complex binding to heparan sulfate proteoglycan in the ECM, resulting in IGF-II-induced signal transduction. This evidence indicates that some ECM-associated growth factors enhance their ability to bind to their receptors by some proteases in the tumor microenvironment. This mechanism of action ('protease-triggered matricrine') represents an attractive model for understanding ECM-tumor interactions.

Immunohistochemical expression of heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) as an angiogenic factor in head and neck tumorigenesis

Heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) is a secreted protein that releases immobilized fibroblast growth factor-2 (FGF-2) from the extracellular matrix and plays a critical role in FGF bioactivation. In the present study co-localization of FGF-2 and HBp17/FGFBP-1 was observed in oral tissues including normal mucosa, hyperplasia, dysplasia of different degrees and oral squamous cell carcinoma (OSCC). The expression score for HBp17/FGFBP-1, FGF-2 as well as vascular endothelial growth factor A (VEGF-A) became higher with the severity of epithelial dysplasia and was highest in severe dysplasia. The expression of HBp17/FGFBP-1, FGF-2 and VEGF-A showed significant association with microvessel density, but no correlation with TNM stages or OSCC recurrence interval. Our results demonstrated that HBp17/FGFBP-1, like VEGF-A and FGF-2, might also promote the induction of tumor angiogenesis. The strongest expression of angiogenic factors in severe dysplasia suggests a potential point for targeting novel anti-angiogenic therapeutic strategies.

Biologic tests for the diagnosis of amniotic fluid embolism

Amniotic fluid embolism is a rare, unpredictable and often lethal complication of pregnancy and childbirth. Because of its variable presentation, an early biologic test would help to establish the diagnosis. We investigated in maternal serum 4 components of amniotic fluid, i.e., alpha-fetoprotein (AFP), l'insuline like growth factor binding protein-1 (IGFBP-1), fetal fibronectin (fFN) and placental alpha1-microglobulin (PAMG-1). On the 6 cesareans controls involved, none of the makers increased after membranes section. PAMG-1 is unsuitable because its detection is always positive or doubtful even in the baseline. On the 7 cases suspected of amniotic fluid embolism, no detectable increase in any of those markers was noted in 3 cases, which is not in favour of this diagnosis. In the remaining cases, IGFBP-1 and fFN became detectable, confirming histological evidences of amniotic fluid embolism in 2 cases. The follow up of those markers in maternal blood confirmed the suspicion of amniotic fluid embolism at 21 wg in one case of ongoing pregnancy. These preliminary results point out the potential interest to assay maternal serum AFP, IGFBP-1 and fFN to confirm amniotic fluid embolism using rapid laboratory tests.

Plasma C‐peptide, insulin‐like growth factor‐I, insulin‐like growth factor binding proteins and risk of colorectal cancer in a nested case‐control study: The Japan public health center‐based prospective study

The physical inactivity and obesity involved in hyperglycemia and hyperinsulinemia is supposed to lead to an increased bioavailability of insulin-like growth factor-I (IGF-I). The carcinogenic effect of IGF-I may be influenced by IGF binding proteins. We investigated the association between plasma levels of C-peptide, a surrogate biomarker of insulin, IGFBP-1, IGF-I or IGFBP-3, and the risk of colorectal cancer in a nested case-control study. During an 11.5-year follow-up, 375 newly diagnosed colorectal cancers were identified in a cohort of 38,373 adults who had returned the baseline questionnaire and provided blood samples. Two matched-controls for each case were selected from the cohort. The odds ratio (OR) of colorectal cancer for plasma levels of each protein was estimated using the conditional logistic regression model adjusted for potential confounding factors. We observed a statistically significant association of plasma C-peptide with colorectal cancer only in men. The ORs were 1.0, 2.3, 2.8 and 3.2 along with quartiles (p trend, 0.0072). The association was stronger in colon cancer (p trend, 0.025) than in rectal cancer (p trend, 0.24). Other peptides were not associated with the risk in either men or women. The results did not change when repeatedly analyzed by tumor invasion levels, tumor sites or follow-up periods. In conclusion, a higher plasma C-peptide may indicate a subsequent risk of colorectal cancer in Japanese men.

Up‐Regulation of Host Cell Genes during Interferon‐γ–Induced PersistentChlamydia pneumoniaeInfection in HL Cells

As a step toward understanding the role played by host gene expression in the development and pathogenesis of persistent Chlamydia pneumoniae infection, modulation of the host-cell transcriptional response during interferon (IFN)- gamma -induced persistent C. pneumoniae infection of HL cells was examined by a cDNA array and then selectively by a real-time quantitative reverse transcription-polymerase chain reaction. We identified 9 host cell genes whose transcription was consistently altered during IFN- gamma -induced persistent C. pneumoniae infection. The strongest up-regulation of persistent infection, compared with controls (active infection and IFN- gamma ) was identified for insulin-like growth factor-binding protein 6, IFN-stimulated protein 15 kDa, cyclin D1, and interleukin-7 receptor genes. These results suggest that, during persistent infection, C. pneumoniae reprograms the host transcriptional machinery that regulates a variety of cellular processes, including adhesion, regulation of the cell cycle, growth, and inflammatory response, all of which might play important roles in the pathogenesis of persistent C. pneumoniae infection.

E. coli에서 발현된 human HtrA1 단백질의 정제와 HtrA1의 serine protease 활성 조건에 관한 연구

E. coli HtrA (High temperature requirement protein A)의 human homologue 중 하나인 HtrA1은 IGFBP를 절단하여 IGF의 활동을 조절하는 serine protease으로 알려졌다. HtrA1의 serine protease 활성이 여러 질병의 발병 mechanism과 연관성을 가진 것으로 예상되고 있지만, 이런 상관관계를 밝히기 위해서 기본적으로 필요한 다량의 HtrA1 단백질의 발현 및 정제조건과 HtrA1 serine protease의 최적 활성조건이 확립되어 있지 않은 상황이다. 따라서 본 연구에서는 pGEX 시스템을 이용하여 E. coli에서 mature HtrA1인 <TEX>${\Delta}149(WT)$</TEX>와 catalytic site mutant인 <TEX>${\Delta}149(S328A)$</TEX>를 85%의 순도로 1 liter 배양 시, 정제된 단백질을 각각 <TEX>$400{\mu}g,\;520{\mu}g$</TEX> 얻을 수 있는 발현조건을 정립하였다. 또한 HtrA1 serine protease 활성은 protease의 농도와 substrate와의 반응시간에 dependent하며, substrate와의 반응온도가 <TEX>$42^{\circ}C$</TEX>일 때 최적의 serine protease활성을 나타내는 것을 알 수 있었다. 특히 <TEX>$200{\mu}M$</TEX>의 HtrA1 serine protease를 <TEX>$37^{\circ}C$</TEX>에서 3시간 반응 시켰을 때, substrate로 사용한 <TEX>${\beta}-casein$</TEX>의 약 50%가 절단되는 것을 관찰하였다. 따라서 이 반응조건에 사용한 HtrA1의 양을 1 unit으로 하여 HtrA1의 serine protease활성을 여러 조건에서 비교 분석할 수 있다 본 연구에서 정립한 mature HtrA1을 다량으로 얻을 수 있는 발헌 및 정제조건과 serine protease 최적 활성조건은 HtrA1의 serine protease 활성과 생물학적 기능의 상관관계를 이해하는데 활용될 수 있을 것이다. Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low <TEX>$460{\mu}g$</TEX> for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was <TEX>$42^{\circ}C$</TEX> instead of <TEX>$37^{\circ}C$</TEX>. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of <TEX>$5{\mu}M\;of\;{\beta}-casein$</TEX> during 3 hr incubation at <TEX>$37^{\circ}C$</TEX>. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.

Short‐term suppression of elevated growth hormone concentrations following insulin‐like growth factor 1 administration in young adults with type 1 diabetes does not alter glomerular filtration or albumin excretion rates

Young adults with type 1 diabetes mellitus (T1DM) have increased glomerular filtration rate (GFR), which may mediate progressive renal disease and microalbuminuria. This may be secondary to low concentrations of insulin-like growth factor (IGF)-I and GH hypersecretion. We tested the hypothesis that restoration of circulating IGF-I concentrations in young adults with T1DM might suppress GH secretion, GFR and urinary albumin excretion. In a randomized double blind crossover study six young adults with T1DM (three men, 19-24 years) received 7 days treatment with rhIGF-I/insulin-like growth factor binding protein (IGFBP)-3 complex (SomatoKine) 0.4 mg/kg/day and placebo. Subjects underwent overnight insulin infusion for euglycaemia, followed by determination of GFR and albumin excretion rate. Following IGF-I/IGFBP-3 complex, overnight insulin requirements (0.15 vs placebo 0.21 mU/kg/min, P < 0.04), plasma insulin (77 vs placebo 152 pmol/l, P < 0.01) and mean overnight GH (2.6 vs placebo 4.8 mU/l, P < 0.04) fell. IGF-I (492 vs placebo 218 ng/ml, P < 0.01) and IGFBP-3 (4.5 vs placebo 3.9 microg/ml, P < 0.05) increased. GFR did not change (145.5 (23.9) ml/min/1.73 m(2) post-IGF-I/IGFBP-3 complex vs 152.2 (19.8) post placebo). Albumin excretion rate did not change 9.5 (5.5-16.6)mg/24 h pre- vs 11.5 (9.9-20.2) post-IGF-I/IGFBP-3 complex and 10.7 (8.1-21.2) pre- vs 11.5 (8.7-29.9) post placebo. Plasma creatinine levels were lower following IGF-I/IGFBP-3 complex (mean +/- SD, 56.2 +/- 16.8 micromol/l) vs placebo (61.5, 45.0, P < 0.02). Seven days treatment with IGF-I/IGFBP-3 complex enhanced overnight insulin sensitivity and reduced GH levels, but there was no effect on glomerular hyperfiltration or albumin excretion rates.

P-806: The importance of insulin like growth factor-1 and insulin like growth factor binding protein-1 during the treatment of polycystic ovary syndrome with clomiphene citrate

The aim of the present study was to define the importance of insulin like growth factor-1 (IGF-1) and insulin like growth factor binding protein-1 (IGFBP-1) during the treatment of Polycystic Ovary Syndrome (PCOS) with Clomiphene Citrate (CC) and the overall effects on the ovulation induction results.