We have developed a novel method for <em>growth</em> <em>factor</em> analysis using a commercial color ink jet printer to fabricate substrata patterned with <em>growth</em> <em>factors</em>. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) spotted in <em>16</em> different areas in varying combinations and concentrations (<em>growth</em> <em>factor</em> array). With this <em>growth</em> <em>factor</em> array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of <em>growth</em> <em>factor</em> array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of <em>growth</em> <em>factors</em> and attachment <em>factors</em> affecting cell <em>growth</em> and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.

Anterior cruciate ligament (ACL) rupture is a common injury often necessitating surgical treatment with graft reconstruction. Due to limitations associated with current graft options, there is interest in a tissue-engineered substitute for use in ACL regeneration. While they represent an important step in translation to clinical practice, relatively few in vivo studies have been performed to evaluate tissue-engineered ACL grafts. In the present study, we immobilized heparin onto electrospun polycaprolactone scaffolds as a means of incorporating basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) onto the scaffold. In vitro, we demonstrated that human foreskin <em>fibroblasts</em> (HFFs) cultured on bFGF-coated scaffolds had significantly greater cell proliferation. In vivo, we implanted electrospun polycaprolactone grafts with and without bFGF into athymic rat knees. We analyzed the regenerated ACL using histological methods up to <em>16</em> weeks post-implantation. Hematoxylin and eosin staining demonstrated infiltration of the grafts with cells, and picrosirius red staining demonstrated aligned collagen fibers. At <em>16</em> weeks postop, mechanical testing of the grafts demonstrated that the grafts had approximately 30% the maximum load to failure of the native ACL. However, there were no significant differences observed between the graft groups with or without heparin-immobilized bFGF. While this study demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture, it also demonstrates that in vitro results do not always predict what will occur in vivo.

OBJECTIVE

To assess <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2/bFGF), which is important in the development and maintenance of the normal prostate and in the development of human benign prostatic hyperplasia (BPH) and prostatic carcinoma, in an animal model of experimentally induced diabetes. Materials and methods Using Western blotting and immunohistochemical analyses, the expression of FGF2 in prostates from several groups of rats was investigated. Rats had diabetes for 8 or <em>16</em> weeks (induced by intravenous injection with 65 mg/kg streptozotocin); rats were also treated with insulin (starting 8 weeks after the induction of diabetes, for 8 weeks), and two further groups acted as age-matched control rats. Immunohistochemical markers for smooth muscle (alpha-actin) and epithelium (cytokeratin) were used to distinguish different cell types in adjacent prostatic sections.

RESULTS

Diabetic rats had smaller prostates and lower serum testosterone levels than their controls; insulin treatment of diabetic rats increased prostatic size and testosterone levels. As shown by Western blotting, diabetes caused greater FGF2 expression than in controls, whereas reverse-transcriptase polymerase chain reaction studies showed similar levels of prostatic FGF-2 mRNA in all groups. Immuno-histochemical studies showed that FGF-2 was expressed in both stromal and epithelial components of the rat prostate. Furthermore, although the expression of FGF2 was higher in epithelial than stromal cells in control prostates, it was distributed uniformly in the diabetic prostate.

CONCLUSIONS

The differences in the level of expression and pattern of distribution of FGF2 suggests a potential role for FGF2 in the changes observed in prostatic growth in diabetic rats.

OBJECTIVE

Previous studies have shown that basic fibroblast growth factor protects against excitatory amino acid toxicity in cultured hippocampal, striatal, and cerebellar neurons. In the current study, we examined the neuroprotective effects of this growth factor on cerebrocortical neurons, which are commonly involved in thromboembolic stroke.

METHODS

Dissociated neuron-glia cultures of embryonic rat cerebral cortex (12 days in vitro) were preincubated with basic fibroblast growth factor (0.1 to 100 ng/mL) for 6 hours before incubation with glutamate (0 to 1000 mumol/L) for 16 hours. The number of phase-bright neurons was taken as an index of neuronal survival.

RESULTS

Basic fibroblast growth factor protected neurons against glutamate toxicity, especially at lower (10, 25, and 50 mumol/L), but not higher (100 and 1000 mumol/L), glutamate concentrations. Neuroprotection was seen at growth factor doses as low as 1 ng/mL.

CONCLUSIONS

Basic fibroblast growth factor protects cultured cerebrocortical neurons against glutamate neurotoxicity.

The mammalian skull vault consists of several membrane bones with different origins. A pair of frontal bones, which occupies the anterior part of the skull vault, is derived from cranial neural crest cells. The frontal bone primordium develops at the superciliary ridge region, then expands towards the top of the head. In this study, we investigated the <em>growth</em> pattern of the frontal bone primordium and the <em>factors</em> involved in this process. In situ hybridization of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 and Runx2, which are markers of preosteoblast, revealed that the frontal bone primordium appears around embryonic day 12.5 (E 12.5) and actively grows between E 14 and E <em>16</em>. We labelled the head mesenchyme of E 13 at the superciliary ridge with DiI by ex-utero surgery and found that the labelled cells were present at the apical edge of the developing frontal bone on E 18. To elucidate the molecular basis of this formation, we cultured E 15 calvarium in the presence of recombinant human bone morphogenetic proteins (rhBMPs); rhBMP-2 and rhBMP-7, but not rhBMP-4, accelerated the <em>growth</em> of the frontal bone domain. These results suggest that the frontal bone primordium grows intrinsically by expanding the primordium not by recruiting surrounding cells, and Bmp-2 and -7 play roles in stimulating the <em>growth</em> of the frontal bone primordium.

OBJECTIVE

Human umbilical cord blood (UCB) is an important source of haematopoietic stem cells; however, the behaviour of progenitor cells obtained from premature and full-term neonates is still a controversy subject. Thus, the aim of this study was to evaluate cell cycle parameters and the proliferative capacity of UCB progenitor cells from premature and full-term neonates.

METHODS

Clonogenic assays were performed with methylcellulose, medium supplemented with recombinant stimulating growth factors and the colonies were scored on the seventh day and the 14th day of culture. A cell cycle study was carried out by DNA analysis using flow cytometry and 30 000 events were acquired; p107 and p130 expressions were analysed by Western blotting.

RESULTS

Cultures obtained from UCB of premature neonates showed an early growth of colony-forming unit (CFU)-burst forming unit erythroid/CFU-granulocyte, erythrocyte, macrophage and megakaryocyte (BFU-E/GEMM), and CFU-granulocyte, macrophage (GM) by the seventh day of culture (P < 0.001). Therefore, the number and morphological characteristics of these colonies were comparable with those obtained from full-term neonates, on the 14th day of culture. At the 14th day, a large amount of CFU-GM was detected in the premature group (P < 0.0032). The premature culture on the 14th day showed fibroblasts and was comparable to those of full-term neonates on the 21st day in terms of number and morphology of the colonies. DNA analysis showed that the number of cells in S-phase was also higher in premature samples when compared to full-term neonates, P < 0.0021 (0 h = 12.8 vs. 2.5%; 16 h = 10.5 vs. 5.9%; 20 h = 13.5 vs. 10.3%; 24 h = 13.8 vs. 9.1%; 48 h = 14.0 vs. 5.4%; 72 h = 20.5 vs. 8.9%; 96 h = 13.8 vs. 7.7%). The Western blotting results demonstrated that p107 and p130 cell cycle protein expressions were higher in premature cells than in full-term cells.

CONCLUSIONS

These results suggest that the higher capacity of proliferation and early differentiation of premature UCB might not be related only to the amount of stem/progenitor cells but also to a different timing of cell cycle entry.

Time-dependent changes in both nerve <em>growth</em> <em>factor</em> (NGF) and acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) levels in the rat brain after cortical cavity lesioning were examined, by using sensitive enzyme immunoassays (EIA) specific for each <em>factor</em>. In the cavity fluid, the NGF level increased rapidly and temporarily with a sharp peak <em>16</em> h after lesioning. A relatively high level was sustained during the next 3-6 days. Contrary to NGF, aFGF was first detectable only 10 days after lesioning, and its level increased gradually until 30 days. These results suggest that NGF and aFGF would play some roles for neuronal repair in different ways.

There is mounting evidence to suggest that stromal cells play an integral role in the progression of prostate cancer (PCa). One of the most frequently altered growth factors in PCa is fibroblast growth factor-2 (FGF-2). It has previously been proposed that early stages of PCa are characterized by a primarily exogenous, that is, stromal cell-derived FGF-2 production, whereas advanced tumors rely more on an autocrine FGF-2 production. Prostate cancer progression is characterized by an increase of genomic instability including aneuploidy and structural chromosomal alterations. Herein, we address 2 problems that have not been comprehensively answered. First, we ask whether exogenous FGF-2 can directly drive genomic instability to promote PCa progression. Second, we investigate whether and to what extent stromal FGF-2 expression is maintained in advanced PCa and whether this influences tumor progression and patient prognosis.

In vitro experiments to investigate the role of FGF-2 in numerical and structural chromosomal instability were performed using immunofluorescence microscopy, fluorescence in situ hybridization and single cell electrophoresis. A human patient-derived xenograft mouse model recapitulating osteoblastic PCa bone metastasis was used for in vivo validation experiments. The prognostic role of stromal FGF-2 expression was analyzed using immunohistochemical staining of a tissue microarray with primary tumor specimens from 162 predominantly high-risk patients with PCa.

Our results show that FGF-2 not only rapidly induces mitotic defects and numerical chromosomal imbalances but also an enhanced DNA breakage to promote chromosomal instability. Using the patient-derived xenograft model, we show that a deregulation of the FGF axis results in an increase of mitotic aberrations as well as DNA damage checkpoint activation in vivo. The FGFR inhibitor dovitinib was found to reduce numerical chromosomal instability as well as DNA breakage, thus underscoring the relevance of the FGF axis in promoting genomic instability. An overexpression of tumor cell-associated FGF-2 was detected in 52 of 162 patients (32.1%), whereas a stromal overexpression was found in 27 of 165 patients (16%). Remarkably, a strong stromal FGF-2 expression was associated with a significantly higher clinical stage and higher biochemical recurrence rate. Patients with strong stromal FGF-2 expression also had a significantly worse biochemical recurrence-free survival.

Our results underscore that exogenous FGF-2 can shape PCa cell genomes and that stromal FGF-2 expression is detectable in a sizeable proportion of advanced PCa where it is associated with adverse clinico-pathological features. Our results highlight the impact of the tumor stroma on malignant progression and provide a rationale for a further exploration of components of the tumor stroma as therapeutic targets in PCa.

The developmental ability and gene expression pattern at 8- to <em>16</em>-cell and blastocyst stages of buffalo (Bubalus bubalis) nuclear transfer (NT) embryos from fetal <em>fibroblasts</em> (FFs), amnion mesenchymal stem cells (AMSCs) and in vitro fertilized (IVF) embryos were compared in the present studies. The in vitro expanded buffalo FFs showed a typical "S" shape <em>growth</em> curve with a doubling time of 41.4 h and stained positive for vimentin. The in vitro cultured undifferentiated AMSCs showed a doubling time of 39.5 h and stained positive for alkaline phosphatase, and these cells also showed expression of pluripotency markers (OCT 4, SOX 2, NANOG), and mesenchymal stem cell markers (CD29, CD44) and were negative for haematopoietic marker (CD34) genes at different passages. Further, when AMSCs were exposed to corresponding induction conditions, these cells differentiated into adipogenic, chondrogenic and osteogenic lineages which were confirmed through oil red O, alcian blue and alizarin staining, respectively. Donor cells at 3-4 passage were employed for NT. The cleavage rate was significantly (P < 0.05) higher in IVF than in FF-NT and AMSC-NT embryos (82.6 ± 8.2 vs. 64.6 ± 1.3 and 72.3 ± 2.2 %, respectively). However, blastocyst rates in IVF and AMSC-NT embryos (30.6 ± 2.7 and 28.9 ± 3.1 %) did not differ and were significantly (P < 0.05) higher than FF-NT (19.5 ± 1.8 %). Total cell number did not show significant (P>> 0.05) differences between IVF and AMSC-NT embryos (186.7 ± 4.2, 171.2 ± 3.8, respectively) but were significantly (P < 0.05) higher than that from FF-NT (151.3 ± 4.1). 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), metabolism (GLUT1) and oxidative stress (MnSOD) regulation were observed in cloned embryos. The transcripts or expression patterns in AMSC-NT embryos more closely followed that of the in vitro derived embryos compared with FF-NT embryos. The results demonstrate that multipotent amnion MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned buffalo embryos.

BACKGROUND

Pin-tract infections are the most common complications of external fixation. To solve the problem, we developed a fibroblast growth factor-2 (FGF-2)-apatite composite layer for coating titanium screws. The purpose of this study was to elucidate the mechanism of the improvement in infection resistance associated with FGF-2-apatite composite layers.

METHODS

We analyzed FGF-2 release from the FGF-2-apatite composite layer and the mitogenic activity of the FGF-2-apatite composite layer. We evaluated time-dependent development of macroscopic pin-tract infection around uncoated titanium control screws (n = 10). Screws coated with the apatite layer (n = 16) and FGF-2-apatite composite layer (n = 16) were percutaneously implanted for 4 weeks in the medial proximal tibia in rabbits.

RESULTS

A FGF-2-apatite composite layer coated on the screws led to the retention of the mitogenic activity of FGF-2. FGF-2 was released from the FGF-2-apatite composite layer in vitro for at least 4 days, which corresponds to a period when 30% of pin-tract infections develop macroscopically in the percutaneous implantation of uncoated titanium control screws. The macroscopic infection rate increased with time, reaching a plateau of 80-90% within 12 days. This value remained unchanged until 4 weeks after implantation. The screws coated with an FGF-2-apatite composite layer showed a significantly higher wound healing rate than those coated with an apatite layer (31.25 vs. 6.25%, p < 0.05). The interfacial soft tissue that bonded to the FGF-2-apatite composite layer is a Sharpey's fiber-like tissue, where collagen fibers are inclined at angles from 30 to 40° to the screw surface. The Sharpey's Wber-like tissue is rich in blood vessels and directly bonds to the FGF-2-apatite composite layer via a thin cell monolayer (0.8-1.7 μm thick).

CONCLUSIONS

It is suggested that the enhanced wound healing associated with the formation of Sharpey's fiber-like tissue triggered by FGF-2 released from the FGF-2-apatite composite layer leads to the reduction in the pin-tract inflammation rate.

The present study was carried out in order to examine the molecular status of selected <em>growth</em> <em>factor</em> receptors (GFR) in urinary bladder lesions, recently described by our group as representing 'Chernobyl cystitis'. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3), epidermal <em>growth</em> <em>factor</em> receptor 1 (EGFR1), EGFR2neu (a member of the same family), p53 and Raf-1 serine/threonine kinase expression were evaluated immunohistochemically in urinary bladder biopsies from 22 men with benign prostate hyperplasia (group 1). For comparison, <em>16</em> men with benign prostate hyperplasia and five women with chronic cystitis living in non-radio-contaminated areas of the country were also investigated as controls (group 2). Additionally, 14 patients with dysplasia, carcinoma in situ (CIS) and primary urothelial carcinoma (UC) operated before the Chernobyl accident as well as 23 patients with UC living in the radio-contaminated areas were included as pre- and post-Chernobyl UC groups 1 and 2, respectively. Chronic proliferative atypical cystitis ('Chernobyl cystitis') was observed in group 1 patients. Foci of dysplasia and CIS were found in 22 (100%) and 19 (86%) of the 22 cases, respectively; moreover, two small UC were also detected. Elevated levels of FGFR3, EGFR2/neu, p53 and to a lesser extent EGFR1 and Raf-1 expression in the urothelial dysplasia and CIS were evident for patients of group 1. Statistically significant differences in immunohistochemical scores for FGFR3, EGFR1, p53 and Raf-1 were observed between groups 1 and 2 and between group 1 and the post-Chernobyl UC group 2, where a change in expression of EGFR2/neu was also noted. A significant decrease in FGFR3 expression in additional pre-Chernobyl UC group 1 with dysplasia, CIS and UC compared with group 1 Chernobyl cystitis cases was detected. Our findings suggest that FGFR and EGFR signaling pathways, associated with p53 and Raf-1 activation, may contribute to multistage urothelial carcinogenesis caused by irradiation, through autocrine or paracrine <em>growth</em> stimulation.

Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) has suppressive effects on food intake. In the present study, the effect of aFGF fragments on food intake were investigated in rats. Infusion of a carboxyl-terminal fragment of aFGF, aFGF-(114-140), did not affect food intake, whereas an amino-terminal fragment of aFGF, aFGF-(1-15), was significantly inhibitory. Other amino-terminal fragments, aFGF-(1-20), aFGF-(1-29) and aFGF-(9-29), did not affect food intake. However, [Ala<em>16</em>]aFGF-(1-29) and [Ser<em>16</em>]aFGF-(1-29) in which the cysteine residue at position <em>16</em> was replaced with alanine and serine, respectively, had significant suppressive effects on food intake. Infusion of a functional antagonist for FGF receptor, anti-FGFR-1 antibody, into the lateral hypothalamus (LHA) significantly increased food intake. The results suggest that: the amino-terminal portion of aFGF is active in food intake suppression; the replacement of cysteine residue by alanine or serine is important in some amino-terminal aFGF fragments; and the LHA is involved in feeding suppression actions by aFGF and some fragments.

We investigated the serum concentration of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and transforming <em>growth</em> <em>factor</em> beta1 (TGFbeta1), using an enzyme linked immunosorbent assay (ELISA) in a group of 18 chronic lymphocytic leukemia (CLL) patients, before and after a successful treatment with cladribine (2-chlorodeoxyadenosine, 2-CdA) and <em>16</em> healthy volunteers. The serum level of bFGF was found to be significantly lower in the control group (median 0.15 pg/ml, range 0.0-15.7 pg/ml), when compared to the untreated CLL patients (median 41.4 pg/ml, range 2.1-292.6 pg/ml) (p=0.0002). After a successful 2-CdA treatment we observed a significantly lower level of this cytokine (median 10.55 pg/ml, range 0.4-140.4 pg/ml) (p=0.0019) in the same patients. However, the level of bFGF in this group was still higher than in the control group (p=0.003). The levels of TGFbeta1 were higher in the group of untreated CLL patients (median 31.36 ng/ml, range 14.36-75.71 ng/ml) than in the control group (median 28.35 ng/ml, range 10.85-70.10 ng/ml) (p=0.029). After the 2-CdA treatment serum concentration of this cytokine decreased significantly (median 20.34 ng/ml, range 3.02-43.85 ng/ml) (p=0.031) with similar levels present to that of the healthy control group (p=0.3). In conclusion, we have shown that the serum concentration of bFGF and TGFbeta1 in CLL patients were significantly reduced after 2-CdA chemotherapy that resulted in remission. The level of these <em>factors</em> might correlate with the activity of the disease.

Fibroblast growth factor (FGF)19 and FGF21 are secreted by the intestine and liver in response to macronutrient intake. Intestinal resection and reconstruction via bariatric surgery may alter their regulation.

We tested the hypothesis that weight loss induced by Roux-en-Y gastric bypass (RYGB) surgery, but not matched weight loss induced by laparoscopic adjustable gastric banding (LAGB), increases postprandial plasma FGF19 and FGF21 concentrations.

Glucose kinetics and plasma FGF19 and FGF21 responses to mixed meal ingestion and to glucose-insulin infusion during a hyperinsulinemic-euglycemic clamp procedure, with stable isotope tracer methods, were evaluated in 28 adults with obesity before and after 20% weight loss induced by RYGB (n = 16) or LAGB (n = 12).

LAGB- and RYGB-induced weight loss increased postprandial plasma FGF19 concentrations (P < 0.05). However, weight loss after RYGB, but not LAGB, increased postprandial plasma FGF21 concentrations (1875 ± 330 to 2976 ± 682 vs 2150 ± 310 and 1572 ± 265 pg/mL × 6 hours, respectively). The increase in plasma FGF21 occurred ∼2 hours after the peak in delivery of ingested glucose into systemic circulation. Glucose-insulin infusion increased plasma FGF21, but not FGF19, concentrations. The increase in plasma FGF21 during glucose-insulin infusion was greater after than before weight loss in both surgery groups without a difference between groups, whereas plasma FGF19 was not affected by either procedure.

RYGB-induced weight loss has unique effects on postprandial FGF21 metabolism, presumably due to rapid delivery of ingested macronutrients to the small intestine and delivery of glucose to the liver.

The amino acid sequence of MG<em>16</em>0, a membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, is more than 90% identical with CFR, a <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) binding protein of chicken membranes, and with ESL-1, a ligand for E-selectin of plasma membranes of myeloid cells; furthermore, MG<em>16</em>0, isolated by immunoaffinity chromatography from rat brain membranes, binds to basic FGF. The gene for MG<em>16</em>0 has been assigned to human chromosome <em>16</em>q22-23. To characterize this protein further in the human, its cDNA was cloned and sequenced. The protein has a large luminal domain composed of an initial proline-glutamine-rich segment, encoded by an uninterrupted exonic sequence of several CAG-CAA repeats. Expansion of CAG repeats has been implicated in the etiology of several neurodegenerative diseases. The proline-glutamine-rich segment is followed by <em>16</em> cysteine-rich repeats that contain five potential asparagine-linked glycosylation sites, which are conserved in the human, rat, mouse, and chicken. The large intralumenal domain of the protein is followed by a single transmembrane domain and a 13-amino-acid cytoplasmic carboxy-terminal tail, which is identical to that in the chicken, rat, and mouse. The overall amino acid identifies between MG<em>16</em>0, CFR, and ESL-1 range from 88% to 95%. In several human fetal and adult tissues, three mRNA transcripts for MG<em>16</em>0 of 10 kb, 5 kb, and 3.8 kb were identified by Northern blot analysis of poly(A)-selected mRNAs. These transcripts may represent alternatively spliced mRNAs of the protein or mRNAs encoding closely related proteins of the Golgi apparatus and/or plasma membranes.

BACKGROUND

Basic fibroblast growth factor (bFGF) promotes vascular repair and angiogenesis and can induce in vitro tissue factor (TF), a potent agent initiating thrombogenesis, which probably plays a role in angiogenesis. We investigated whether bFGF administration induced TF expression by monocytes and vascular cells.

RESULTS

We studied TF expression in normally fed (n=16) and cholesterol-fed (2% for 6 weeks, n=16) rabbits. Animals were then randomized to receive intravenous bFGF (2.5 microg twice weekly for 3 weeks) or saline injections. TF expression was evaluated in mononuclear cells from arterial blood and in aortic sections by an immunohistochemical assay using a monoclonal anti-rabbit TF antibody (activator protein 1). Monocyte TF expression was increased by bFGF administration in both normal and hypercholesterolemic rabbits (129+/-45 versus 19+/-3 mU TF/1000 monocytes, P<0.05, and 31+/-12 versus 7+/-1 mU TF/1000 monocytes, P<0.005, respectively) and was further increased by stimulation of monocytes by endotoxin in vitro. TF expression was lower in hypercholesterolemic rabbits than in normal rabbits. In the media of the vascular wall, bFGF induced strong TF expression in normal rabbits and only weak TF expression in hypercholesterolemic ones.

CONCLUSIONS

This study demonstrates that systemic administration of bFGF induces an impressive increase of TF expression in circulating monocytes and in the vascular wall in normal and to a lower extent in hypercholesterolemic rabbits. The significance of this observation in terms of inducing thrombosis in vivo needs clarification.

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.