Loss of renal phosphate wasting in a child with autosomal dominant hypophosphatemic rickets caused by a FGF23 mutation.

A girl with autosomal dominant hypophosphatemic rickets, presented with clinical, radiological and laboratory signs of rickets at the age of 11 months. She showed a good response to the treatment with low doses of oral phosphate and calcitriol. Surprisingly, she lost her renal phosphate wasting at the age of 8 years, indicating that the disturbed phosphate metabolism can be compensated by hormonal or other factors.     

A mutation in the fibroblast growth factor 14 gene is associated with autosomal dominant cerebellar ataxia [corrected

Hereditary spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative disorders for which >/=14 different genetic loci have been identified. In some SCA types, expanded tri- or pentanucleotide repeats have been identified, and the length of these expansions correlates with the age at onset and with the severity of the clinical phenotype. In several other SCA types, no genetic defect has yet been identified. We describe a large, three-generation family with early-onset tremor, dyskinesia, and slowly progressive cerebellar ataxia, not associated with any of the known SCA loci, and a mutation in the fibroblast growth factor 14 (FGF14) gene on chromosome 13q34. Our observations are in accordance with the occurrence of ataxia and paroxysmal dyskinesia in Fgf14-knockout mice. As indicated by protein modeling, the amino acid change from phenylalanine to serine at position 145 is predicted to reduce the stability of the protein. The present FGF14 mutation represents a novel gene defect involved in the neurodegeneration of cerebellum and basal ganglia.     

Acidic fibroblast growth factor enhances peripheral nerve regeneration in vivo

When added to a collagen-filled nerve guide, purified acidic fibroblast growth factor (aFGF) increased the number of myelinated axons that regenerated across a 5-mm nerve gap distance. In addition, a greater number of primary sensory and motor neurons extended axons through the nerve guide in animals treated with aFGF. Thus the effect of aFGF on peripheral nerve regeneration is not simply an increase in axonal branching within the nerve guide tube. This is the first highly purified growth factor since nerve growth factor that has been shown to promote nerve regeneration in vivo. This experimental model provides a convenient and quantitative means to assess the effects of putative neuronotropic factors on peripheral nerve regeneration in vivo.     

In vivo biological potency of rat and human growth hormone-releasing factor and fragments of human growth hormone-releasing factor

The biological potency of the synthetic replicates of three peptides isolated from a human pancreatic tumor with growth hormone releasing activity and rat hypothalamic growth hormone-releasing factor was evaluated in conscious freely-moving rats and anesthetized rats. All 4 peptides are equipotent on a molar basis in their ability to stimulate GH secretion. Studies with synthetic fragments of the human derived material indicated that the amino-terminal amino acid is required for activity. Deletion of as many as 13 amino acids from the carboxy-terminal failed to decrease GH-releasing activity; however, deletion of 16 amino acids resulted in a significant decrease and deletion of 20 amino acids resulted in complete loss of bioactivity.     

The fibroblast growth factor family: Structural and biological properties

This article sumarizes the structural and biological properties of the family of fibroblast growth factors (FGF). Basic FGF (bFGF) and acidic FGF (aFGF) are the best characterized members of this family. bFGF and aFGF are potent modulators of cell proliferation, motility and differentiation. They are also potent angiogenesis factors in vivo. Some of the important biological characteristics of bFGF and aFGF discussed in the review include the affinity of bFGF and aFGF for heparin, their lack of secretion in culture and their association with extracellular matrix. Recently, several oncogenes, 40–50% homologous in sequence to bFGF and aFGF have been identified. These include int-2, hst, K-fgf and FGF-5. The structural and biological properties of these FGF-related oncogenes are also discussed.     

The R1947X mutation of NF1 causing autosomal dominant neurofibromatosis type 1 in a Chinese family

Neurofibromatosis type 1 is a common autosomal dominant disorder with a high rate of penetrance. It is caused by the mutation of the tumor suppressor gene NF1, which encodes neurofibromin. The main function of neurofibromin is down-regulating the biological activity of the proto-oncoprotein Ras by acting as a Ras-specific GTPase activating protein. In this study, we identified a Chinese family affected with neurofibromatosis type 1. The known gene NF1 associated with NF1 was studied by linkage analysis and by direct sequencing of the entire coding region and exon-intron boundaries of the NF1 gene. The R1947X mutation of NF1 was identified, which was co-segregated with affected individuals in the Chinese family, but not present in unaffected family members. This is the first report, which states that the R1947X mutation of NF1 may be one of reasons for neurofibromatosis type 1 in Chinese population.     

Basic fibroblast growth factor enhances the osteogenic differentiation induced by bone morphogenetic protein-6 in vitro and in vivo

Some members of the bone morphogenetic protein subfamily (BMP-2 and -7) are currently used in orthopedic surgery for several applications. Although their use is considered safe at short term, the high doses of growth factors needed make these treatments expensive and their safety uncertain at long term. BMP-6 has been much less studied than BMP-2 and -7, but some authors suggest that this BMP might have a stronger osteogenic activity than the previously mentioned. Having in mind that angiogenesis plays a well-known role during bone formation, the aim of this work was to study the effect of combining BMP-6 with bFGF on both the growth and differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs), as well as on in vivo osteogenesis. We demonstrate that a low dose of bFGF enhances the osteogenic differentiation of MSCs induced by BMP-6 in vitro. Furthermore, we also demonstrate that bone formation in vivo induced by BMP-6 can be accelerated and enhanced by adding a low dose of bFGF, what might suggest a synergic effect between these growth factors on in vivo osteogenesis.     

Monoclonal antibodies directed against basic fibroblast growth factor which inhibit its biological activity in vitro and in vivo

A panel of four murine monoclonal IgG1 antibodies (mAbs) to a recombinant form of basic fibroblast growth factor (bFGF) was produced using somatic cell fusion techniques. Non-linear regression analysis of radioimmunoassay data for each mAb yielded the following dissociation constants (nM) for their interactions with bFGF: DE6 (0.822); AF11 (2.0); FE8 (2.31); and DG2 (20.0). One of the mAbs, DG2, was identified as a bFGF neutralizing antibody on the basis of its ability to inhibit, in vitro, the binding of [125I]-bFGF to high and low affinity bFGF sites on cultured baby hamster kidney cells and bFGF-induced [3H]-thymidine incorporation in cultured 3T3 cells, and in vivo, the angiogenic response to bFGF in a rat kidney capsule model of angiogenesis. The other mAbs displayed varying inhibitory activities in these assays. These mAbs, particularly DG2, may be well suited for a number of applications in bFGF research including immunoassays, immunohistochemical studies, and as functional antagonists of bFGF for examining its role in physiological processes such as reproduction, growth, and development.     

NH2-terminal cleavage of xenopus fibroblast growth factor 3 is necessary for optimal biological activity and receptor binding.

Fibroblast growth factor 3 (FGF3) was originally identified as the mouse proto-oncogene Int-2, which is activated by proviral insertion in tumors induced by mouse mammary tumor virus. To facilitate the biological characterization of the ligand, we have analyzed its homologue in Xenopus laevis, XFGF3. Here we confirm that the X. laevis genome contains two distinct FGF3 alleles, neither of which is capable of encoding the NH2-terminally extended forms specified by the mouse and human FGF3 genes. Unlike the mammalian proteins, XFGF3 is efficiently secreted as a Mr 31,000 glycoprotein, gp31, which undergoes proteolytic cleavage to produce an NH2-terminally truncated product, gp27. Processing removes a segment of 18 amino acids immediately distal to the signal peptide that is not present in the mammalian homologues. By inserting an epitope-tag adjacent to the cleavage site, we show that a substantial amount of the gp27 is generated intracellularly, although processing can also occur in the extracellular matrix. Two residues are also removed from the COOH terminus. To compare the biological properties of the different forms, cDNAs were constructed that selectively give rise to the larger, gp31, or smaller, gp27, forms of XFGF3. As judged by their ability to cause morphological transformation of NIH3T3 cells, their mitogenicity on specific cell types, and their affinity for the IIIb and IIIc isoforms of Xenopus FGF receptors, gp27 has a much higher biological activity than gp31. Sequence comparison revealed an intriguing similar cleavage motif immediately downstream of the signal peptide cleavage site in the NH2-terminus of mouse and human FGF3. Analysis of secreted mutant mouse FGF3 confirmed an additional NH2-terminal processing at the corresponding sequence motif. NH2-terminal trimming of Xenopus and mammalian FGF3s may therefore be a prerequisite of optimal biological activity.     

STAT1 hyperphosphorylation and defective IL12R/IL23R signaling underlie defective immunity in autosomal dominant chronic mucocutaneous candidiasis

We recently reported the genetic cause of autosomal dominant chronic mucocutaneous candidiasis (AD-CMC) as a mutation in the STAT1 gene. In the present study we show that STAT1 Arg274Trp mutations in the coiled-coil (CC) domain is the genetic cause of AD-CMC in three families of patients. Cloning and transfection experiments demonstrate that mutated STAT1 inhibits IL12R/IL-23R signaling, with hyperphosphorylation of STAT1 as the likely underlying molecular mechanism. Inhibition of signaling through the receptors for IL-12 and IL-23 leads to strongly diminished Th1/Th17 responses and hence to increased susceptibility to fungal infections. The challenge for the future is to translate this knowledge into novel strategies for the treatment of this severe immunodeficiency.     

Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa

Protein conformational disorders, which include certain types of retinitis pigmentosa, are a set of inherited human diseases in which mutant proteins are misfolded and often aggregated. Many opsin mutants associated with retinitis pigmentosa, the most common being P23H, are misfolded and retained within the cell. Here, we describe a pharmacological chaperone, 11-cis-7-ring retinal, that quantitatively induces the in vivo folding of P23H-opsin. The rescued protein forms pigment, acquires mature glycosylation, and is transported to the cell surface. Additionally, we determined the temperature stability of the rescued protein as well as the reactivity of the retinal-opsin Schiff base to hydroxylamine. Our study unveils novel properties of P23H-opsin and its interaction with the chromophore. These properties suggest that 11-cis-7-ring retinal may be a useful therapeutic agent for the rescue of P23H-opsin and the prevention of retinal degeneration.     

Klotho ablation converts the biochemical and skeletal alterations in FGF23 (R176Q) transgenic mice to a Klotho-deficient phenotype

Transgenic mice overexpressing fibroblast growth factor (FGF23) (R176Q) (F(Tg)) exhibit biochemical {hypophosphatemia, phosphaturia, abnormal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] metabolism} and skeletal (rickets and osteomalacia) abnormalities attributable to FGF23 action. In vitro studies now implicate the aging-related factor Klotho in the signaling mechanism of FGF23. In this study, we used a mouse genetic approach to validate in vivo the pivotal role of Klotho in the metabolic and skeletal derangements associated with FGF23 (R176Q) overexpression. To this end, we crossed mice heterozygous for the hypomorphic Klotho allele (Kl(+/-)) to F(Tg) mice and obtained F(Tg) transgenic mice homozygous for the Kl-hypomorphic allele (F(Tg)/Kl(-/-)). Mice were killed on postnatal day 50, and serum and tissues were procured for analysis and comparison with F(Tg), wild-type, and Kl(-/-) controls. From 4 wk onward, F(Tg)/Kl(-/-) mice were clearly distinguishable from F(Tg) mice and exhibited a striking phenotypic resemblance to the Kl(-/-) controls. Serum analysis for calcium, phosphorus, parathyroid hormone, 1,25(OH)(2)D(3), and alkaline phosphatase activity confirmed the biochemical similarity between the F(Tg)/Kl(-/-) and Kl(-/-) mice and their distinctness from the F(Tg) controls. The characteristic skeletal changes associated with FGF23 (R176Q) overexpression were also dramatically reversed by the absence of Klotho. Hence the wide, unmineralized growth plates and the osteomalacic abnormalities apparent in trabecular and cortical bone were completely reversed in the F(Tg)/Kl(-/-) mice. Nevertheless, independent actions of Klotho on bone were suggested as manifested by alterations in mineralized bone, and in cortical bone volume which were observed in both the Kl(-/-) and F(Tr)/Kl(-/-) mutants. In summary, our findings substantiate in vivo the essential role of Klotho in the mechanism of action of FGF23 in view of the fact that Klotho ablation converts the biochemical and skeletal manifestations resulting from FGF23 overexpression to a phenotype consistent with Klotho deficiency.     

Inhibitors of epidermal growth factor receptor tyrosine kinase: optimisation of potency and in vivo pharmacokinetics

The structure-activity and structure-property relationships of anilinoquinazoline inhibitors of EGFR were investigated. Strategies to lower volume of distribution and shorten half-life through structure and pKa modulation are discussed.     

The discovery of basic fibroblast growth factor/fibroblast growth factor-2 and its role in haematological malignancies

Basic fibroblast growth factor/fibroblast growth factor-2 is one of the best characterized of the pro-angiogenic cytokines. This review describes its history, as well as its role in tumor angiogenesis associated with haematological malignancies, as traced by the main contributions to the international medical literature.     

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells.     

The role of fibroblast growth factor 8 in cartilage development and disease

Fibroblast growth factor 8 (FGF‐8), also known as androgen‐induced growth factor (AIGF), is presumed to be a potent mitogenic cytokine that plays important roles in early embryonic development, brain formation and limb development. In the bone environment, FGF‐8 produced or received by chondrocyte precursor cells binds to fibroblast growth factor receptor (FGFR), causing different levels of activation of downstream signalling pathways, such as phospholipase C gamma (PLCγ)/Ca²⁺, RAS/mitogen‐activated protein kinase‐extracellular regulated protein kinases (RAS/MAPK‐MEK‐ERK), and Wnt‐β‐catenin‐Axin2 signalling, and ultimately controlling chondrocyte proliferation, differentiation, cell survival and migration. However, the molecular mechanism of FGF‐8 in normal or pathological cartilage remains unclear, and thus, FGF‐8 represents a novel exploratory target for studies of chondrocyte development and cartilage disease progression. In this review, studies assessing the relationship between FGF‐8 and chondrocytes that have been published in the past 5 years are systematically summarized to determine the probable mechanism and physiological effect of FGF‐8 on chondrocytes. Based on the existing research results, a therapeutic regimen targeting FGF‐8 is proposed to explore the possibility of treating chondrocyte‐related diseases.