06-106 Sigma-AldrichAnti-IGFBP-1 Antibody

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler's murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.

Studies on the mechanisms of decidualization and endometriosis are often hampered by lack of primary endometrial cells. To facilitate in vitro studies, we established a human endometrial stromal cell line, KC02-44D, immortalized with human telomerase reverse transcriptase. Upon exposure to ovarian stimuli, KC02-44D cells showed similar cytoskeletal marker or gene expression and biochemical phenotype to primary endometrial stromal cells. KC02-44D would be useful for studies of human endometrial function and its associated pathologies.

We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP (c-FLIPL), but not the short form (c-FLIPS), enhanced adhesion and motility. Downregulation of c-FLIPL with siRNA decreased phosphorylation of FAK and ERK, while overexpression of c-FLIPL increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed c-FLIPL-promoted motility. Inhibition of ROCK by Y27632 suppressed the c-FLIPL-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of c-FLIPL increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced c-FLIPL- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the c-FLIPL-promoted cell motility. We conclude that c-FLIPL promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.

We report the localization of insulin-like growth factor I (IGF-I) and a 25-kDa form of insulin-like growth factor-binding protein (IGF-BP-1) in adult rat kidney. The antigens were localized using a rabbit anti-human IGF-I antibody, and a rabbit anti-human IGF-BP-1 antibody raised against human 25-kDa IGF-BP-1 purified from amniotic fluid. Immunohistochemistry by the avidin-biotin peroxidase conjugate technique showed that both peptides are located in the same nephron segments, in the same cell types. The most intense staining was in papillary collecting ducts. There was moderate staining also in cortical collecting ducts and medullary thick ascending limbs of Henle's loop. In collecting ducts the antigens were shown to be present in principal cells but not in intercalated cells. In distal convoluted tubules, cortical thick ascending limbs, and in structures presumptively identified as thin limbs of Henle's loops there was only modest staining. The macula densa, however, lacked immunoreactivity. Colocalization of IGF-I and IGF-BP-1 in the same cells supports the notion, derived from studies on cultured cells, that the actions of IGF-I may be modified by IGF-BPs that are present in the same location.

Human amniotic fluid has been shown to contain a protein that binds insulin-like growth factor I and II (IGF-I and IGF-II). Partially purified preparations of this protein have been reported to inhibit the biologic actions of the IGFs. In these studies our laboratory has used a modified purification procedure to obtain a homogeneous preparation of this protein as determined by polyacrylamide gel electrophoresis and amino acid sequence analysis. During purification the ion exchange chromatography step resulted in two peaks of material with IGF binding activity termed peaks B and C. Each peak was purified separately to homogeneity. Both peaks were estimated to be 31,000 daltons by polyacrylamide gel electrophoresis and their amino acid compositions were nearly identical. Amino acid sequence analysis showed that both peaks had identical N-terminal sequences through the first 28 residues. Neither protein had detectable carbohydrate side chains and each had a similar affinity for radiolabeled IGF-I (1.7-2.2 x 10(10) liters/mol). In contrast, these two forms had marked differences in bioactivity. Concentrations of peak C material between 2 and 20 ng/ml inhibited IGF-I stimulation of [3H]thymidine incorporation into smooth muscle cell DNA. In contrast, when peak B (100 ng/ml) was incubated with IGF-I there was a 4.4-fold enhancement of stimulation of DNA synthesis. Additionally, pure peak B was shown to adhere to cell surfaces, whereas peak C was not adherent. The non-adherent peak C inhibited IGF-I binding to its receptor and to adherent peak B. We conclude that human amniotic fluid contains two forms of IGF binding protein that have very similar physiochemical characteristics but markedly different biologic actions. Since both have similar if not identical amino acid compositions, N-terminal sequences, and do not contain carbohydrate, we conclude that they differ in some other as yet undefined post-translational modification.

Insulin-like growth factor I (IGF-I) is a peptide growth factor that circulates bound to carrier proteins. One form of carrier protein (mol wt, approximately 26K) is not believed to be GH dependent, is relatively unsaturated, and modulates the cellular response to IGF-I. This study was undertaken to determine the variables that control the plasma concentration of this protein, which was measured using a specific RIA. The mean plasma 26K IGF-binding protein (IGF-BP) concentration in 15 normal fasting subjects at 0800 h was 9.4 +/- 4.4 (+/- SD) micrograms/L. The mean value in GH-deficient patients was increased to 19.5 +/- 10.1 micrograms/L (n = 60; P less than 0.05), and it was 7.3 +/- 4.3 micrograms/L in patients with acromegaly (n = 31). The GH dependency of these changes is further supported by the observation that subjects who received GH injections had a 51% reduction in their fasting values. Nutritional intake appeared to be a more important controlling variable than GH. During an overnight fast plasma 26K IGF-BP values increased approximately 4-fold in 6 normal subjects. After 2 days of fasting, the mean value in 7 obese subjects rose progressively from 6.5 +/- 2.3 to 11.7 +/- 5.4 micrograms/L (P less than 0.001), and it increased further to 19.2 +/- 5.9 micrograms/L by day 4 of fasting; after 2 days of refeeding it returned to the prefasting level of 6.8 +/- 1.9 micrograms/L. Likewise, ingestion of a standard test meal resulted in a significant decrease in mean plasma 26K IGF-BP from a fasting value of 8.4 +/- 2.9 to 5.6 +/- 2.8 micrograms/L 4 h postprandially (P less than 0.05). In summary, the plasma concentrations of the 26K IGF-I-BP fluctuate widely in response to dietary manipulation, whereas GH status appears to be a secondary controlling variable.