OBJECTIVE

Given recent physiological and in situ hybridization evidence for the presence of a water channel in corneal epithelium, this study was conducted to investigate its expression and characteristics using cultured bovine corneal epithelial cells (CBCEPCs).

METHODS

CBCEPCs were grown in DMEM containing 2 ng/ml fibroblast growth factor and 6% fetal bovine serum. To determine their osmotic permeability (Pf), cells were passaged onto rectangular glass coverslips, and anisotonically induced volume changes were monitored by light scattering. To investigate expression, poly(A+) RNA from CBCEPCs was injected into Xenopus laevis oocytes, and the Pf of the oocytes was determined.

RESULTS

For CBCEPCs challenged with a 10% hypotonic solution at 37 degrees C, the kinetic constant of volume change was k=0.52+/-0.04 seconds(-1), and the calculated Pf 72+/-6 microm/sec (n=16). The Pf of oocytes injected with water was 14+/-1.8 microm/sec (n=4); injection with poly(A+) RNA from CBCEPCs increased Pf to 77+/-6 microm/sec (n=6). This increase in Pf was inhibited by 72% (reduced to 22+/-1 microm/sec) by 0.3 mM HgCl2 and was inhibited by 56% to 58% by coinjection with aquaporin (AQP)5 antisense oligonucleotide.

CONCLUSIONS

The comparatively high Pf determined for CBCEPCs, the presence of mRNA encoding water channels, and sensitivity to mercurial agents are typical of the expression of functional water channels. The predominant message is for AQP5, although the evidence was consistent with the presence of additional water channels. These findings bring renewed support for the notion that the epithelium can contribute to corneal hydration homeostasis.

OBJECTIVE

In this study, the authors evaluated the efficacy of a new surgical strategy for reconnecting the injured brachial plexus with the spinal cord using fibrin glue containing acidic fibroblast growth factor as an adhesive and neurotrophic agent.

METHODS

Eighteen patients with preganglionic brachial plexus injuries, each with varying degrees of upper limb dysfunction, underwent cervical laminectomy with or without sural nerve grafting. The treatment of each avulsed root varied according to the severity of the injury. Some patients also underwent a second-stage operation involving supraclavicular brachial plexus exploration for reconnection with the corresponding segment of cervical spinal cord at the trunk level. Muscle strength was graded both pre- and postoperatively with the British Medical Research Council scale, and the results were analyzed with the Friedman and Wilcoxon signed-rank tests.

RESULTS

Muscle strength improvements were observed in 16 of the 18 patients after 24 months of follow-up. Significant improvements in mean muscle strength were observed in patients from all repair method groups at 12 and 24 months postoperatively (p < 0.05). Statistical significance was not reached in the groups with insufficient numbers of cases.

CONCLUSIONS

The authors' new surgical strategy yielded clinical improvement in muscle strength after preganglionic brachial plexus injury, such that nerve regeneration may have taken place. Reconnection of the brachial plexus to the cervical spinal cord is possible. Functional motor recovery, observed through increases in Medical Research Council-rated muscle strength in the affected arm, is likewise possible.

Abnormal gene expression in somatic cell nuclear transfer embryos due to aberrant epigenetic modifications of the donor nucleus may account for much of the observed diminished viability and developmental abnormalities. The present study compared the developmentally important gene expression pattern at 4-cell, 8- to <em>16</em>-cell, morula, and blastocyst stages of buffalo nuclear transfer (NT) embryos from adult <em>fibroblasts</em> (AFs) and amniotic fluid stem cells (AFSCs). In vitro fertilized embryos were used as control embryos. Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), <em>growth</em> <em>factor</em> signaling, and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), oxidative stress (MnSOD), metabolism (GLUT1) regulation were observed in cloned embryos. The expression of transcripts in AFSC-NT embryos more closely followed that of the in vitro fertilized embryos compared with AF-NT embryos. It is concluded that AFSCs with a relatively undifferentiated genome may serve as suitable donors which could be reprogrammed more efficiently to reactivate expression of early embryonic genes in buffalo NT.

The <em>growth</em>-promoting effects of pituitary <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and "chondrocyte <em>growth</em> <em>factor</em>" (CGF), a contaminant of NIH-bovine TSH and ovine LH, were studied in monolayer and/or organ culture of epiphyseal plate chondrocytes of rabbits 1 day to 10 weeks old. The response to FGF (50 ng/ml) and CGF(TSH) and CGF(LH) (64 micrograms/ml) was age-dependent. In cultures from animals aged 4 weeks or older, the <em>growth</em> <em>factors</em> consistently stimulated DNA synthesis while decreasing incorporation of radiosulfate into matrix macromolecules. In organ cultures of the <em>growth</em> plate of rabbits less than 1 week old, FGF and NIH-TSH and LH actually diminished rather than promoted incorporation of 3H-thymidine. In organ culture the generation times of newborn rabbit proliferating zone chondrocytes, measured in vivo and in vitro by nuclear grain count dilution in 3H-thymidine autoradiographys, were 11 and <em>16</em> to 17 hours respectively. FGF and LH increased the generation time of 1- and 4-day old rabbit chondrocytes from <em>16</em> to over 24 hours. The stimulatory effect of CGF(TSH) in the older age group was much greater than that of NIH(LH). The dog-dose response curves of FGF and CGF(LH) were parallel, supporting Jones and Addison's view that the CGF activity of CGF(LH) derives from its content of FGF.

The surface ganglioside marker A2B5 was originally detected on neurons, but has subsequently been shown to be expressed on a wide range of macroglial and non-neural cells. This marker has been used in vitro to categorize subpopulations of neural cells within the central nervous system, as well as defining developmental pathways of macroglia. These categorizations are based on the assumption that this marker cannot easily be modulated. In this study of cerebellar cultures we demonstrate that A2B5 can be induced on approximately <em>16</em>% of A2B5 non-expressing cells by the <em>growth</em> <em>factors</em>: basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (b-FGF); acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (a-FGF); and, to a lesser degree, epidermal <em>growth</em> <em>factor</em> (EGF).

After the detection of epidermal <em>growth</em> <em>factor</em> and transforming <em>growth</em> <em>factor</em> alpha in various body fluids and human saliva the current study aimed to investigate the presence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in human saliva. Basic FGF is stimulating the proliferation of cells of mesodermal and neuroectodermal origin and is highly angiogenetic. After ELISA technique was established, saliva was collected from eight healthy individuals. Run in duplicate, 14 (87.5%) of the <em>16</em> samples investigated contained measurable amounts of bFGF. In the samples containing bFGF the concentration varied between 0.1 pg/mL and 8.4 pg/mL (mean concentration, 3.8 pg/mL; SD, 3.5). There was no correlation between age and sex and bFGF concentrations. It is therefore concluded that bFGF is present in human saliva and may even constitute a constant component. The physiological importance of this finding is discussed.

Tumor-promoting phorbol esters, like <em>growth</em> <em>factors</em>, elicit pleiotropic responses involving biochemical pathways that lead to different biological responses. Genetic variant cell lines that are resistant to mitogenic, differentiation, or transformation responses to tumor promoters have been valuable tools for understanding the molecular bases of these responses. Studies using the mouse epidermal JB6 cell lines that are sensitive or resistant to tumor promoter-induced transformation have yielded new understanding of genetic and signal transduction events involved in neoplastic transformation. The isolation and characterization of cloned mouse promotion sensitivity genes pro-1 and pro-2 is reviewed. A new activity of pro-1 has been identified: when transfected into human cancer prone basal cell nevus syndrome <em>fibroblasts</em> but not normal <em>fibroblasts</em> mouse pro-1 confers lifespan extension on these cells. Recently, we have found that a pro-1 homolog from a library of nasopharyngeal carcinoma, but not the homolog from a normal human library, is activated for transferring promotion sensitivity. The many genetic variants for responses to tumor promoters have also proved valuable for signal transduction studies. JB6 P- cells fail to show the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced synthesis of two proteins of 15 and <em>16</em> kD seen in P+ cells. P-, P+, and TPA transformed cells show a progressive decrease in both basal and TPA-inducible levels of a protein kinase C substrate of 80 kD. P- cells are relatively resistant both to anchorage-independent transformation and to a protein band shift induced by the calcium analog lanthanum. It appears that one or more calcium-binding proteins and one or more pro genes may be critical determinants of tumor promoter-induced neoplastic transformation.

Dopamine-releasing cells derived from embryonic stem cells (ESCs) are potentially valuable in cell transplantation therapy for Parkinson's disease. There have been many recent investigations of the induction of dopamine-releasing cells from mouse and primate ESCs. However, there are major obstacles to application of dopamine-releasing ESC progeny to cell transplantation therapy, including host immune responses to transplanted cells and the difficulty of collecting dopamine-releasing cells from culture dishes undamaged. To overcome these obstacles, in the present study, cynomolgus monkey ES cell (cESC) aggregates enclosed in agarose microcapsules were cultured in 3 kinds of media: Glasgow minimum essential medium-based medium (GBM); GBM-containing conditioned medium of PA6 cells; and GBM supplemented with <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)8, sonic hedgehog, and ascorbic acid (GBM(+)) under free-floating culture conditions. Of these 3 culture media, GBM(+) most efficiently induced dopamine-releasing cells. Addition of FGF8, sonic hedgehog, and ascorbic acid to the culture medium during culture days 10 to 15, days 12 to 15, and days <em>16</em> to 20, respectively, facilitated the generation of dopamine-releasing cells. Because various characteristics of cESCs are reported to be similar to those of human ESCs, we expect that the study using cESCs will provide useful information for cell transplantation therapy of Parkinson's disease.

BACKGROUND

To investigate the changes in angiogenic growth factor expression before and after gefitinib treatment, and the association between this expression and response to gefitinib treatment, we measured circulating levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), matrix metalloproteinase (MMP) -2 and -9, and tissue inhibitors of metalloproteinase (TIMP) -1 and -2 in patients with non-small cell lung cancer (NSCLC).

METHODS

Serum and plasma samples were collected from 52 patients before and after gefitinib treatment. The levels of VEGF, bFGF, MMP-2, MMP-9, TIMP-1 and TIMP-2 were measured using a sandwich enzyme immunoassay kit.

RESULTS

Of the 52 patients, 17 (32.7%) achieved a partial response, 19 (36.5%) had stable disease and 16 (30.8%) had progressive disease. The levels of VEGF, bFGF, MMP-2, MMP-9, TIMP-1 and TIMP-2 did not change significantly after gefitinib treatment, even in responders. The levels of VEGF in volunteers, responders and non-responders were 384 +/- 86.4, 404 +/- 94.3 and 719 +/- 99.8 pg/ml, respectively. The difference between volunteers and responders was not significant (P = 0.540), while the differences between volunteers and non-responders (P = 0.031), and responders and non-responders (P = 0.028) were significant.

CONCLUSIONS

Although our results indicate that gefitinib treatment does not affect circulating levels of angiogenic growth factors even in patients who showed a response to gefitinib treatment, low levels of VEGF may predict response to gefitinib treatment in patients with NSCLC.

Abnormal wound healing processes can result in hypertrophic scars and keloids. Transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) and hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> (HGF/SF) are biphasic <em>growth</em> <em>factor</em> cytokines in physiologic and pathophysiologic conditions. Findings have shown TGF-beta1 to be pivotal in the formation of keloid tissue. Therefore, neutralizing antibodies may allow wound healing without keloid formation. As reported, TGF-beta1 is antagonized by HGF/SF. Some authors have reported that exogenous administration of HGF/SF prevented scar formation. Hence, this study targeted TGF-beta1 and determined the levels of HGF/SF in <em>fibroblast</em> cell culture. Keloid tissue was taken from seven patients. Another seven patients with mature nonhypertrophic scar served as controls. All tissues were cultured, and <em>fibroblast</em> cultures were used for further experiments. The TGF-beta1 antisense was administered at 3 and 6 micromol/ml, and HGF/SF levels were determined after <em>16</em>, 24, and 48 h of incubation. The levels of HGF/SF showed significant differences after incubation with antisense oligonucleotides. The increasing antisense levels resulted in increased HGF/SF levels (up to 87.66 pg/ml after 48 h of incubation). In conclusion, targeting TGF-beta1 resulted in significantly increased levels of HGF/SF. The clinical relevance could include the use of locally administered HGF/SF in protein or gene form to minimize formation of keloids. Nevertheless, wound healing is the result of many interacting cytokines, so neutralizing or targeting one protein could result in no significant effect.

The <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are involved in hematopoiesis and tumorigenesis. However, little is known about the contribution of the FGFs identified within the past 10 years to leukemogenesis. To elucidate whether these FGFs (FGF-8, -9, -10, -11, -12, -13, -14, -<em>16</em>, -17, -18, -19, -20, and -21) are expressed in leukemic cells, we performed RT-PCR analyses using 28 cell lines. The members of a fetal-oncogenic subfamily, FGF-8/-17/-18, were often expressed (53.5%, 25.0%, and 32.1%) with the co-expression of their receptors. Realtime quantitative-PCR analysis showed that FGF-8/-17 were aberrantly expressed in patients with acute leukemia. Moreover, cell proliferation assays revealed the proliferation activity of FGF-17 on leukemic cells expressing its receptors. These results demonstrated that certain recently identified FGFs play an important role in the <em>growth</em> of leukemic cells, possibly with an autocrine mode of action, and that these FGFs will become novel biomarkers for hematopoietic tumors.

BACKGROUND

Pleomorphic adenoma (PA) is the most common salivary gland tumour. Although classified as benign, it has a tendency to recur (recurrent pleomorphic adenomas (RPA)), as well as the ability to undergo malignant transformation. It has been suggested that mutations in various families of growth factors and growth factor receptions are involved in the autonomous growth of tumour cells. The aim of the present study was to investigate the participation of platelet-derived growth factor (PDGF)-A, PDGF-B, PDGF-Rα, fibroblast growth factor (FGF)-2, Flg and BEK in PA, RPA and recurrent pleomorphic adenoma with malignant transformation (TRPA).

METHODS

18 cases of PA, 16 cases of RPA and two cases of RPA with focal malignant transformation (TRPA) were analysed for growth factor expression utilising immunohistochemical techniques via tissue microarray.

RESULTS

There was a significant difference in PDGF-A, PDGF-B, PDGF-Rα, FGF-2, Flg and BEK expression in all groups. When comparing non-recurrent with recurrent tumours, PDGF-A, PDGF-B, PDGF-Rα, FGF-2, Flg and BEK reactivity in RPA was stronger than that observed in PA. All proteins were highly expressed in TRPA.

CONCLUSIONS

This research suggests that PDGF-A, PDGF-B, PDGF-Rα, FGF-2, BEK and Flg can be related to the recurrence of PA. In addition, this study shows that TRPA cells overexpress all growth factors, which has been reported in association with the malignant transformation.

Fibroadenoma (FA) is the most common benign tumor of the breast in adult women. Some FA have a highly cellular stroma, making it difficult to differentiate from phyllodes tumors (PT). Forty-three FA were grouped into: (i) 27 conventional type (FACT) median stromal cellularity (SC) of highest cellular area (HCA), < or = 125 cells/1 high-power field (HPF); and (ii) <em>16</em> cellular variant (FACV) median SC of HCA,>> 125 cells/1 HPF. These were studied for the proliferative activity of their stromal cells. Expression of c-fos, p53, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) in the stromal cells were examined in the FA and 12 PT to determine whether it is possible to separate FACV from FACT. The proliferative activity of stromal cells was evaluated by the labeling index (LI) of proliferating cell nuclear antigen (PCNA). Conventional type fibroadenoma stromal cells had the lowest frequency of c-fos, p53, bFGF, FGFR and VEGF protein expression; PT stromal cells had the highest frequency of expression; and FACV stromal cells had an intermediate frequency of expression. Multivariate analysis demonstrated that bFGF and FGFR expression are significantly correlated with SC of FA. Separation of FACV from FACT by SC seems appropriate in revealing the phenotypic and biological differences of FA. The SC of FA seems to be regulated by bFGF and FGFR expression.

Immunoglobulins reactive against basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) were obtained from the serum of a single rabbit immunized against residues [1-24] of bFGF conjugated to keyhole limpet hemocyanin (KLH). Pure immunoglobulin preparations no. 1 and no. 2 were prepared using different affinity chromatography columns and preabsorption to KLH-coupled Sepharose for preparation no. 1. Both preparations no. 1 and no. 2 were specific for bFGF in in vitro assays. Competition with synthetic peptides suggests that preparations no. 1 and no. 2 recognize predominantly epitope(s) within residues [<em>16</em>-24]bFGF or residues [1-10]bFGF, respectively, in situ. Furthermore, no. 2 (but not no. 1) antibodies can react with tissue-(heparin-)-bound antigen. When used in indirect immunofluorescence for bFGF in frozen heart sections, preparation no. 1 stained predominantly muscle intercalated discs (IcDs); muscle nuclei were also stained, in an overall punctate fashion. Preparation no. 2 stained muscle nuclei strongly, in association with the nuclear envelope; it also stained basement-membrane associated bFGF. Differences in immunostaining were also observed in uterine smooth muscle and kidney sections but not in skeletal muscle. It is plausible that accessibility of various epitopes within the amino-terminal region depends strongly on the local interactions of bFGF. Our data illustrate the importance of using several different antibodies to localize bFGF in a tissue.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day <em>16</em>, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology.

BACKGROUND

Chronic inflammation disrupts dental pulp regeneration by disintegrating the recruitment process of progenitors for repair. Bone marrow-derived mesenchymal stem cells (BM-MSCs) share the common features with dental pulp stem cells (DPSCs). The aim of the study was to investigate the migration of BM-MSCs toward DPSCs in response to inflammatory chemoattractants. Additionally, our studies also delineated the signaling mechanisms from BM-MSCs in mediating the proliferation and differentiation of DPSCs in vitro.

METHODS

Human DPSCs and BM-MSCs between passages 2 and 4 were used and were grown in odontogenic differentiation medium. Mineralization was determined by alizarin red staining analysis. Migration was assessed using crystal violet staining in cells grown in Boyden chamber Transwell inserts (Corning Inc Foundation, Tewksbury, MA). The mineralization potential of DPSCs was evaluated using alkaline phosphatase activity assay. Real-time polymerase chain reaction analysis was performed to assess the gene expression profile of chemokine (C-X-C motif) ligand (Cxcl) 3, 5, 6, 10, 11, 12, 14, and <em>16</em>; stromal cell-derived <em>factor</em> (SDF) α; vascular endothelial <em>growth</em> <em>factor</em>; and <em>fibroblast</em> <em>growth</em> <em>factor</em>.

RESULTS

Interferon gamma (FN-γ) treatment significantly abrogated the differentiation potential of DPSCs as shown by using alizarin red and alkaline phosphatase activity analysis. An increase in the migration of BM-MSCs was documented when cocultured with IFN-γ-treated DPSCs. RNA expression studies showed an increase in the levels of Cxcl6 and Cxcl12 in BM-MSCs when cocultured with IFN-γ-treated DPSCs. Additionally, an up-regulation of proangiogenic factors vascular endothelial growth factor and fibroblast growth factor were observed in DPSCs exposed to IFN-γ.

CONCLUSIONS

Our findings indicate that inflamed IFN-γ-treated DPSCs release factors (presumably Cxcl6 and 12) that contribute to the homing of MSCs. This model might provide a potential research tool for studying MSC-DPSC cross talk and for future studies involving the recruitment and sustainability of progenitor stem cells sustaining the inflammatory cascade to treat pulp inflammation.

DNA synthesis was measured <em>16</em> h after stimulation of Swiss 3T3 <em>fibroblasts</em> in the resting phase with various <em>growth</em> <em>factors</em> (platelet-derived <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, lysophosphatidic acid and thrombin). When extracellular Ca2+ was chelated by EGTA, or when the influx of Ca2+ from outside to inside the cell was blocked by cobalt, DNA synthesis was completely inhibited. As there was no effect whatsoever on DNA synthesis when Ca2+ was chelated, or when the influx of Ca2+ was blocked up to the first 4 h after <em>growth</em> stimulation, it was concluded that, at an early stage, Ca2+ influx from outside to inside the cell is not related to the transition from the G1 to the S phase. A Ca2+/calmodulin-dependent protein kinase II inhibitor (KN-62) had no effect on DNA synthesis. However, cyclosporin A and FK-506, which are inhibitors of Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin), markedly inhibited DNA synthesis stimulated by all of the <em>growth</em> <em>factors</em>. These results indicate that calcineurin plays a role, not only in activation of T-cells of the immune system in the initial phase, but also in DNA synthesis in <em>fibroblasts</em>. It was concluded that Ca2+ influx from outside to inside the cell during the mid-to-late G1 phase, followed by calcineurin activation, is essential as a mechanism of <em>growth</em> signal transduction.

As a key humoral regulator of phosphate homeostasis and its involvement in the pathogenesis of human disease, human <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (hFGF23) has become a particularly attractive therapeutic target. To prepare soluble and bioactive recombinant human FGF23 to meet the increasing demand in its pharmacological application, small ubiquitin-related modifier (SUMO)-FGF23 fusion gene and FGF23 non-fusion gene were amplified by standard PCR methods and cloned into vector pET-22b and pET-3c, then transformed into Escherichia coli Rosetta (DE3) and BL21 (DE3). The best combination of plasmid and host strain was screened, and only Rosetta (DE3)/pET-SUMO-FGF23 was screened for rhFGF23 protein expressed. The average bacterial yield and the soluble expression level of recombinant hFGF23 of three batches attained 687 ± 18 g and 30 ± 1.5%, respectively, after treatment with 0.4 mM isopropyl-thio-β-galactopyranoside for 19 h at <em>16</em> °C in a 30-L fermentor, after which it was purified by DEAE Sepharose FF and nickel nitrilotriacetic acid affinity chromatography. Once cleaved by the SUMO protease, the recombinant human FGF23 was released from the fusion protein. The purity of rFGF23 was shown by high performance liquid chromatography to be greater than 90% and the yield was 60 ± 1.5 mg/L. In vitro data showed that the purified rFGF23 can induce the phosphorylation of mitogen-activated protein kinases in the glioma U251 cell. The results of in vivo animal experiments also showed that rFGF23 could decrease the concentration in the plasma of normal rats fed with a fixed formula diet.

An important part of the biosynthesis of proteoglycans is the epimerization of glycosaminoglycan chains. As a consequence of the conversion of chondroitin sulfate (CS) to dermatan sulfate (DS), the glycosaminoglycans become more flexible and enable DS to perform more sophisticated signaling functions. In a recent study, we generated a chimera (S222A) composed of a truncated form of a DS (decorin) and CS (CSF-1) containing proteoglycan and analyzed the influence of the core protein on the extent of epimerization. C-terminal truncation constructs from S222A enabled us to identify an amino acid segment that lies within the CSF-1 part which prevents DS synthesis. Co-localization experiments using S222A-HA and DCN-Flag showed different intracellular localizations for the proteoglycans during biosynthesis. A data base search revealed a sequence motif (TNWVP) within the CSF-1 moiety that is found to be important in other proteoglycans. A single substitution of tryptophan-2<em>16</em> to leucine (W2<em>16</em>L) in the chimera S222A increased the amount of l-IdoA to 12-<em>16</em>%. Co-localization with an ER-marker demonstrated that the biosynthesis of recombinant decorin is similar to the chimera S222A and S222A(W2<em>16</em>L) in HEK293 cells. Co-staining of S222A-HA and S222A(W2<em>16</em>L)-Flag showed different intracellular localizations for the proteoglycans. A more detailed analysis of the glycosaminoglycans reflects a similar total sulfate content for S222A and S222A(W2<em>16</em>L). The 4/6 sulfation ratio was similar for decorin and S222A, but altered for S222A(W2<em>16</em>L). However, the binding of <em>fibroblasts</em> <em>growth</em> <em>factor</em>-1 to CS/DS was only partially dependent on epimerization. These results are consistent with the model in which the core protein, via the amino acid tryptophan, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase.

Icariin (ICA), which is an essential bioactive component extracted from the herb Epimedium, possesses neuroprotective properties. The aim of the present study was to investigate the regulatory roles of ICA in cell proliferation and gene expression in human neural stem cells (NSCs) in vitro. Single cells were isolated from the corpus striatum of <em>16</em>‑20‑week human fetuses obtained following spontaneous abortion. The cells were cultured in Dulbecco's modified Eagle's medium/F12 complete medium and were characterized by immunostaining and cell differentiation assay. NSCs were treated with ICA, and cell proliferation was assessed using the Cell Counting kit‑8 cell proliferation assay kit. In addition, neurosphere formation was comparatively studied between the ICA‑treated and control cells. cDNA microarray analysis was performed to examine the effects of ICA on gene expression. Altered expression of genes important for regulating NSC proliferation was further analyzed by quantitative polymerase chain reaction (qPCR). The results demonstrated that typical neurospheres appeared after 7‑10 days of culturing of individual cells isolated from the corpus striatum. These cells expressed nestin, an important NSC marker, and in the presence of differentiation medium they expressed β‑III‑tubulin, a specific neuronal marker, and glial fibrillary acidic protein, an astrocyte marker. Treatment with ICA enhanced NSC proliferation and the formation of neurospheres. Microarray data and pathway analysis revealed that the genes regulated by ICA were involved in several signaling pathways, including the Wnt and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) pathways, which are important for the regulation of NSC function. Upregulation of frizzled class receptor 7 and dishevelled segment polarity protein 3, which are key players in the Wnt pathway, and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1, which is the receptor for bFGF, and downregulation of glycogen synthase kinase‑3β, which is a Wnt pathway inhibitor, was further validated by qPCR. In conclusion, ICA promoted proliferation and regulated gene expression in human NSCs, thus suggesting that ICA may exert its neuroprotective effects by regulating NSC activity.

δ‑like ligand 4 (DLL4)‑Notch signaling is associated with tumor resistance to anti‑vascular endothelial <em>growth</em> <em>factor</em> (VEGF) therapy. Furthermore, Notch signaling is critical for the maintenance of colon cancer stem cells (CSCs), which are relevant in drug resistance and tumor angiogenesis. CD44 is a transmembrane glycoprotein and is considered a putative marker of CSCs. To assess the association of Notch intracellular cleaved domain (NICD), DLL4 and CD44 expression with the efficacy of anti‑angiogenic drugs, a series of samples derived from patients with advanced colon cancer enrolled in prospective clinical trials were analyzed. Histological samples from 51 primary tumors that originated from patients treated with bevacizumab‑based first‑line chemotherapy were analyzed by immunohistochemistry for NICD, DLL4 and CD44 expression, and CD31 for microvessel count. The expression levels of genes relevant for angiogenesis [angiopoietin (ANGPT)1, ANGPT2, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)1, FGF2, epidermal <em>growth</em> <em>factor</em>, placental <em>growth</em> <em>factor</em>, VEGFA and DLL4] were detected by reverse transcription‑quantitative PCR using RNA extracted from the frozen tissues of four tumors with low and four tumors with high NICD expression. Strong NICD levels were observed in 12/51 (24%) of the patients, whereas <em>16</em>/51 (31%) of the colon cancer subjects exhibited high CD44 expression. Strong CD44 staining was associated with high NICD levels compared with the CD44 expression levels noted in samples with low NICD levels (67 vs. 20%, P=0.005). No association was observed with regards to the expression levels of NICD, CD44 and the other aforementioned biomarkers. High expression levels of NICD and CD44 predicted reduced progression‑free survival (P<0.001) and overall survival (P=0.002). No significant differences in the expression of angiogenesis‑related genes were detected between low and high NICD‑expressing tumors. In conclusion, NICD and CD44 tissue levels exhibited an association and may be related to a reduced survival rate in patients with advanced colon cancer treated with bevacizumab.

The immunosuppressive protein transforming <em>growth</em> <em>factor</em> beta (TGF-beta) inhibits the activation of various immune effector cells including cytotoxic T lymphocytes and may therefore inhibit the efficacy of immunostimulatory interleukin-2 (IL-2) gene therapy. In this study, we investigated the effect of TGF-beta downregulation on IL-2 gene therapy in the intraperitoneal model of murine ovarian teratoma (MOT). MOT cells, like many human ovarian carcinomas, were found to produce TGF-beta. Production of TGF-beta by MOT cells was suppressed using a TGF-beta antisense plasmid vector (pCEP4/TGF-beta antisense). Subcutaneous immunization of C3H mice with a mixture of IL-2 gene-transduced <em>fibroblasts</em> and TGF-beta antisense-modified MOT cells induced significantly better protection against a subsequent intraperitoneal tumor challenge compared with immunization with unmodified MOT cells alone [11/<em>16</em> (69%) vs 4/21 (19%) tumor-free animals, P < 0.01]. Immunization with either a mixture of IL-2 gene modified <em>fibroblasts</em> and unmodified MOT cells [2/12 (17%) tumor-free animals] or TGF-beta antisense-modified MOT cells alone (0/13 tumor free animals) failed to induce significant protection compared with immunization with unmodified MOT cells. These data show that combined TGF-beta antisense and IL-2 gene therapy is required to generate effective antitumor responses in the MOT model. Our findings suggest that tumor cell expression of immunosuppressive <em>factors</em> may inhibit cytokine immunogene therapy and may have potential implications for the development of future clinical immunogene therapy protocols.

Idiopathic pes equinovarus is a congenital deformity of the foot and lower leg defined as a fixation of the foot in adduction, supination, and varus. Although the pathogenesis of clubfoot remains unclear, it has been suggested that <em>fibroblasts</em> and <em>growth</em> <em>factors</em> are involved. To directly analyze the protein composition of the extracellular matrix in contracted tissue of patients with clubfoot. A total of 13 infants with idiopathic clubfoot treated with the Ponseti method were included in the present study. Tissue samples were obtained from patients undergoing surgery for relapsed clubfeet. Contracted tissues were obtained from the medial aspect of the talonavicular joint. Protein was extracted after digestion and delipidation using zip-tip C18. Individual collagenous fractions were detected using a chemiluminescent assay. Amino acid analysis of tissue samples revealed a predominance of collagens, namely collagen types I, III, and VI. The high content of glycine and h-proline suggests a predominance of collagens I and III. A total of 19 extracellular matrix proteins were identified. The major result of the present study was the observation that the extracellular matrix in clubfoot is composed of an additional <em>16</em> proteins, including collagens V, VI, and XII, as well as the previously described collagen types I and III and transforming <em>growth</em> <em>factor</em> β. The characterization of the general protein composition of the extracellular matrix in various regions of clubfoot may help in understanding the pathogenesis of this anomaly and, thus, contribute to the development of more efficacious therapeutic approaches.

1. A pure strain of <em>fibroblasts</em> has been isolated from Sarcoma 10 of the Crocker Foundation. After about <em>16</em> months of life in vitro, the malignancy of the strain is as great as that of the original tumor. 2. The strain has been compared with a strain of normal rat <em>fibroblasts</em>. The malignant cells are generally larger, coarser, and more refringent than normal cells. They possess all the morphological characteristics of <em>fibroblasts</em>. They do not show any abnormalities and never degenerate and die. They are to all appearances healthy cells. Their mode of locomotion is identical with that of normal <em>fibroblasts</em>. Their colonies are larger, but the architecture is similar. 3. The residual activity of both cell types, the duration of their life, and their rate of <em>growth</em> in a nutrient medium are almost identical. 4. The sarcomatous <em>fibroblasts</em> liquefy a rat plasma coagulum while normal <em>fibroblasts</em> do not. They turn phenol red golden yellow whereas, under the same conditions, normal cells turn it pinkish orange. 5. Sarcomatous and normal <em>fibroblasts</em> of the rat multiply to an unlimited degree in chick embryo juice. They live for only a short time in rat serum and chick serum. Calf liver digest will suffice for an unlimited proliferation of sarcoma <em>fibroblasts</em>, but fails to support the life of normal <em>fibroblasts</em> for very long. 6. The presence of bone marrow greatly increases the rate of <em>growth</em> of sarcomatous <em>fibroblasts</em> cultivated in rat serum, while it only slightly affects that of the normal cells. The unlimited <em>growth</em> of the sarcomatous tissue in animals to which it is transplanted may be attributed to the presence of macrophages, which are a normal constituent of the tumor, and possibly are a necessary <em>factor</em> of its <em>growth</em> in vivo.