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Biomedical subjects

G M Fox

Publications and source records attributed to G M Fox.

At least 37 records · Page 2Linked to original sources
PubMed1992

Cysteine to serine substitutions in basic fibroblast growth factor: effect on inclusion body formation and proteolytic susceptibility during in vitro refolding.

We have investigated the effect of cysteine to serine substitutions in human basic fibroblast growth factor (bFGF) on the formation of inclusion bodies in Escherichia coli. Using a temperature-sensitive expression, system, about 30% of human bFGF, which contains four cysteines at positions 26, 70, 88, and 93, is deposited into inclusion bodies. A single mutation at position 88 and a double mutation at positions 70 and 88 do not greatly alter the partition of bFGF into soluble and insoluble cell fractions. However, a single substitution of cysteine 70 by serine decreases the fraction of soluble bFGF significantly. When cysteines 26 and 93 (conserved among related growth factors) are replaced by serines, no soluble bFGF is formed in E. coli. Cysteine to serine substitutions also affect proteolytic susceptibility of bFGF during in vitro refolding from crude inclusion bodies. About 60% of human bFGF is lost to proteolytic degradation during in vitro refolding. Replacement of cysteines by serines increases the total recovery of bFGF, although more aggregates are formed during refolding. Ser-88-bFGF was expressed at the highest level, gave the highest soluble fraction in vivo, and exhibited the greatest fractional recovery and was recovered with the largest insoluble fraction after in vitro refolding. Thermal stability experiments at 42 degrees C and 70 degrees C revealed that cysteine to serine substitutions did not cause aggregation of the folded protein in vitro.

Base Sequence↗
PubMed1992

Platelet-derived growth factor (BB homodimer), transforming growth factor-beta 1, and basic fibroblast growth factor in dermal wound healing. Neovessel and matrix formation and cessation of repair.

Recombinant platelet-derived growth factor (BB homodimer, rPDGF-BB), transforming growth factor beta 1 (rTGF-beta 1), and basic fibroblast growth factor (rbFGF) can accelerate healing of soft tissues. However, little information is available characterizing the components of wound matrix induced by these growth factors and the molecular mechanisms underlying accelerated repair and wound maturation. In this study, the composition, quantity, and rate of extracellular matrix deposition within growth factor-treated lapine ear excisional wounds were analyzed at different stages of healing using specific histochemical and immunohistochemical stains, coupled with image analysis techniques. Single application of optimal concentrations of each growth factor accelerated normal healing by 30% (P less than 0.0003); rPDGF-BB markedly augmented early glycosaminoglycan (GAG) and fibronectin deposition, but induced significantly greater levels of collagen later in the repair process, compared with untreated wounds rTGF-beta 1 treatment led to rapidly enhanced collagen synthesis and maturation, without increased GAG deposition. In contrast, rbFGF treatment induced a predominantly angiogenic response in wounds, with a marked increase in endothelia and neovessels (P less than 0.0001), and increased wound collagenolytic activity (P less than 0.03). rbFGF-treated wounds did not evolve into collagen-containing scars and continued to accumulate only provisional matrix well past wound closure. These results provide new evidence that growth factors influence wound repair via different mechanisms: 1) rPDGF-BB accelerates deposition of provisional wound matrix; 2) rTGF-beta 1 accelerates deposition and maturation of collagen; and 3) rbFGF induces a profound monocellular angiogenic response which may lead to a marked delay in wound maturation, and the possible loss of the normal signal(s) required to stop repair. These results suggest that specific growth factors may selectively regulate components of the repair response by differing mechanisms, offering the potential for targeted therapeutic intervention.

Animals↗
PubMed1991

Delayed-onset pseudophakic endophthalmitis.

We reviewed 19 cases of delayed-onset pseudophakic endophthalmitis in which diagnostic cultures were performed at one month or more after cataract extraction with posterior chamber intraocular lens implantation. We isolated four different organisms in these 19 cases: 12 Propionibacterium species (63%), three Candida parapsilosis (16%), three Staphylococcus epidermidis (16%), and one Corynebacterium species (5%). Because of the unusual delayed-onset features of these cases and the retrospective nature of this study, a variety of treatment regimens were used. Twelve patients had recurrence of marked inflammation despite an apparent initial cure, and ten of these patients had positive culture results on repeat examination of intraocular fluids. Nine patients continued to be treated with topical corticosteroids postoperatively to suppress low-grade inflammation. Of the 19 patients, 16 had final visual acuity of 20/400 or better. Delayed-onset pseudophakic endophthalmitis had a more favorable visual prognosis, compared to acute-onset endophthalmitis.

Adrenal Cortex Hormones↗
PubMed1991

Three-dimensional structures of acidic and basic fibroblast growth factors.

Members of the fibroblast growth factor (FGF) family of proteins stimulate the proliferation and differentiation of a variety of cell types through receptor-mediated pathways. The three-dimensional structures of two members of this family, bovine acidic FGF and human basic FGF, have been crystallographically determined. These structures contain 12 antiparallel beta strands organized into a folding pattern with approximate threefold internal symmetry. Topologically equivalent folds have been previously observed for soybean trypsin inhibitor and interleukins-1 beta and -1 alpha. The locations of sequences implicated in receptor and heparin binding by FGF are presented. These sites include beta-sheet strand 10, which is adjacent to the site of an extended sequence insertion in several oncogene proteins of the FGF family, and which shows sequence conservation among the FGF family and interleukin-1 beta.

Amino Acid Sequence↗
PubMed1991

Detection of herpesvirus DNA in vitreous and aqueous specimens by the polymerase chain reaction.

Members of the herpesvirus family, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV), have been recognized as causal agents of chorioretinal inflammatory diseases. We investigated the use of the polymerase chain reaction for the detection of CMV, HSV, and EBV genomes in aqueous, subretinal fluid, and vitreous specimens in patients with clinically diagnosed CMV retinitis. Cytomegalovirus but not HSV or EBV genomic sequences were detected in all of these clinical specimens. We also investigated 18 normal aqueous and eight normal vitreous specimens obtained from patients undergoing cataract or vitrectomy surgery. Cytomegalovirus, HSV, and EBV DNA were not detected in any of the normal aqueous specimens. There was one weakly positive CMV normal vitreous, but none was HSV or EBV positive by the polymerase chain reaction. These results indicate that the polymerase chain reaction may be useful as a rapid and sensitive diagnostic technique to aid in the confirmation of clinical observations.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Altered distribution of basic fibroblast growth factor in diabetic retinopathy.

Basic fibroblast growth factor (FGF) is a potent endothelial cell mitogen that has been proposed to play a role in proliferative diabetic retinopathy and other neovascular processes. Our understanding of the in vivo role of basic FGF in the pathogenesis of these disorders is limited. We studied the immunolocalization of basic FGF in 16 clinical cases of diabetic retinopathy to determine whether the normal retinal distribution of basic FGF changed during the development of diabetic retinopathy and correlated with the onset of retinal neovascularization. By using monoclonal and affinity-purified polyclonal antibodies against basic FGF and heparan sulfate proteoglycan (HSPG), we found that basic FGF colocalized with HSPG to vascular basement membranes. As the basement membranes thickened during the progression of diabetic retinopathy, the intraretinal stores of immunoreactive basic FGF and HSPG expanded. With the development of neovascularization, the colocalization of basic FGF and HSPG changed; HSPG localized to basement membranes, while basic FGF localized intracellularly, with only minimal basement membrane immunoreactivity. Incubations of the neovascular fronds with exogenous basic FGF demonstrated multiple HSPG glycosaminoglycan-binding sites for basic FGF, indicating that basic FGF had not been released from the matrix of neovascular fronds by heparitanase digestion.

Antibodies, Monoclonal↗
PubMed1990

Alteration in folding efficiency and conformation of recombinant human tumor necrosis factor-alpha by replacing cysteines 69 and 101 with aspartic acid 69 and arginine 101.

An analog of human tumor necrosis factor-alpha (TNF-alpha) was created involving the replacement of Cys69 with Asp and Cys101 with Arg. The solution structure and behavior of this analog were compared with the native protein. The analog exhibited a greatly decreased folding efficiency following dilution from urea, but essentially identical circular dichroic spectra in both the folded and unfolded states. The Stokes radius of the native and analog TNF-alpha in the folded state were identical, with the analog exhibiting a slight broadening of the eluting peak. The fluorescence emission spectrum of the native protein exhibits a plateau from 320 to 328 nm, while the spectrum of the analog consisted of a single peak with a maximum at 335 nm. The analog also had a 1.4-fold increase in the fluorescence intensity. Limited proteolysis of the analog resulted in only one of the two peptides seen following digestion of the native protein, and this product was less stable than the equivalent native protein fragment. The analog exhibited a 10-fold lower cytolytic activity than the native protein. These results demonstrated that the disulfide bond is not necessary for folding and activity, but are consistent with the analog having a looser, more flexible structure in solution than the native TNF-alpha.

Amino Acid Sequence↗
PubMed1990

Cogan's syndrome and seroreactivity to Lyme borreliosis.

We document the case of a young woman with bilateral nonluetic, interstitial keratitis, vestibuloauditory problems, and bilateral recurrent facial palsies, who had strongly positive serologic tests of Lyme borreliosis. To our knowledge, this is the first reported case of positive serologic tests for Borrelia burgdorferi in a patient with Cogan's syndrome.

Adult↗
PubMed1989

Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor.

Using oligo site-directed mutagenesis, we have modified our synthetic gene for human basic fibroblast growth factor (bFGF) to replace all four cysteine codons with serine codons. The corresponding protein was expressed in Escherichia coli and purified from inclusion bodies by solubilization in urea followed by a series of column chromatographies and a folding step. The resulting protein, having no cysteine residues, is unable to form either intramolecular or intermolecular disulfide bonds. The secondary and tertiary structures of the purified analog, as determined by circular dichroism and fluorescence spectroscopy, were identical within experimental error to recombinant bovine and human bFGF with unaltered amino acid sequences. Reflecting the similar conformation, the analog protein exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.

Amino Acid Sequence↗
PubMed1989

v-src genes stimulate neurite outgrowth in pheochromocytoma (PC12) variants unresponsive to neurotrophic factors.

The NGF-nonresponsive rat pheochromocytoma PC12 variant nnr5, isolated by Green et al. (J Cell Biol 102:830-843, 1986), responds poorly or not at all to fibroblast growth factor. Transformation of PC-12nnr5 cells with v-src-expressing retroviruses results in vigorous neurite outgrowth, similar to that seen in the parent cell line. Thus though the PC12nnr5 cell line has a greatly impaired ability to respond to neurotrophic factors it still may extend neurites. This data is consistent with a model in which PC12nnr5 cells are unable to propagate intracellular second messengers, and this defect may be related to the expression of c-src gene products.

Animals↗
PubMed1988

Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine.

We have chemically synthesized the gene encoding bovine basic fibroblast growth factor (bFGF) and cloned it into a plasmid vector. This gene was then used as a template for site-directed mutagenesis to produce the human bFGF gene and a gene coding for an analog in which serine residues were substituted for the cysteine residues at positions 70 and 88. All three constructs were cloned and expressed in Escherichia coli and the proteins purified. The recombinant human and bovine bFGFs exhibited the potent mitogenic activity toward both fibroblasts and endothelial cells, which characterizes natural bFGF. The serine-70,88 analog and natural sequence bovine and human forms were equally active in all assays. Sulfhydryl titration of the purified recombinant bovine bFGF in 4.8 M guanidine hydrochloride indicated the presence of approximately two free sulfhydryl groups. This was consistent with the sequence analysis of peptides derived from trypsin digestion, which suggests that cysteines 70 and 88 exist in free sulfhydryl form while cysteines 26 and 93 form a disulfide bond. Circular dichroism shows that the protein has little ordered structure but is folded into a rigid tertiary configuration. Carboxymethylation of the free sulfhydryl groups resulted in no change in the mitogenic activity or conformation. These results are consistent with previous suggestions that, for tissue-derived bFGF, at least 2 of the 4 cysteines in the molecule are not involved in a disulfide bond.

Amino Acid Sequence↗
PubMed1987

Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis.

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.

Animals↗
PubMed1987

Experimental autoimmune uveoretinitis (EAU) induced by retinal interphotoreceptor retinoid-binding protein (IRBP): differences between EAU induced by IRBP and by S-antigen.

Rats immunized with the retinal interphotoreceptor retinoid-binding protein (IRBP) develop an inflammatory eye disease, "experimental autoimmune uveoretinitis" (EAU). The ocular changes which characterize the EAU induced by IRBP resemble those seen in rats which develop EAU by immunization with another retinal protein, S-antigen (S-Ag). Yet, the two antigens do not cross-react antigenically and the two diseases differ by several features: At low doses (less than or equal to 4 micrograms/rat), IRBP was more uveitogenic in Lewis rats than was S-Ag, inducing disease more reproducibly and with earlier onset time. On the other hand, at higher doses (greater than or equal to 20 micrograms/rat) the disease induced by S-Ag was more severe than that induced by the same doses of IRBP. Rats of various inbred strains differed in their susceptibility to EAU induced by these two antigens. In particular, BN rats were more susceptible to IRBP-induced EAU than to the S-Ag-induced disease, while WF and RCS-rdy+ rats developed severe EAU when immunized with S-Ag but showed minimal or no ocular change when immunized with IRBP.

Animals↗
PubMed1986

Monkey erythropoietin gene: cloning, expression and comparison with the human erythropoietin gene.

The erythropoietin (Epo) gene from Cynomolgus monkeys has been isolated from a kidney cDNA library using mixed 20-mer oligodeoxynucleotide probes. The gene encodes a 168 amino acid (aa) mature protein with a calculated Mr of 18,490 and a presumptive signal peptide of 24 aa. The Epo gene, when transfected into Chinese hamster ovary (CHO) cells, produces a glycosylated protein with an apparent Mr of 34,000. The expressed product is biologically active in vivo. The monkey gene exhibits 92% and 94% homology to the human gene at the aa and nucleotide sequence levels, respectively. When compared with the human Epo, monkey Epo has an additional 3-aa residue at the N terminus of the mature protein and a deletion of an internal lysine residue.

Amino Acid Sequence↗
PubMed1985

Cloning and expression of the human erythropoietin gene.

The human erythropoietin gene has been isolated from a genomic phage library by using mixed 20-mer and 17-mer oligonucleotide probes. The entire coding region of the gene is contained in a 5.4-kilobase HindIII-BamHI fragment. The gene contains four intervening sequences (1562 base pairs) and five exons (582 base pairs). It encodes a 27-amino acid signal peptide and a 166-amino acid mature protein with a calculated Mr of 18,399. The erythropoietin gene, when introduced into Chinese hamster ovary cells, produces erythropoietin that is biologically active in vitro and in vivo.

Amino Acid Sequence↗
PubMed1983

Species-specific homogeneity of the primate Alu family of repeated DNA sequences.

We have determined the base sequence of several cloned Alu family members from the DNAs of a new world monkey (owl monkey) and a prosimian (galago). The three owl monkey Alu family members reported here belong to a single 300 base pair consensus sequence which closely resembles the human Alu family consensus. The galago Alu family members can best be represented as belonging to either of two related but distinct consensus sequences. One of the two galago Alu family subgroups (Type I) more accurately resembles the human consensus sequence than does the other subgroup (Type II). In this work we compare base sequences of human and galago Type I Alu family members. There are several examples of species-specific differences between the human and Type I galago sequences indicating that the Alu family members are effectively homogenized within a species.

Animals↗
PubMed1981

A complex repeated DNA sequence within the Drosophila transposable element copia.

A 320 nucleotide repeated DNA sequence within the copia coding element of Drosophila melanogaster has been identified and characterized. This sequence has been localized by DNA-DNA hybridization and electron microscopic analysis of heteroduplexes to the approximate middle of the 5 kb copia coding region. The primary sequence of this repeated DNA has been determined. The sequence is composed of three related subunits, 35-37 nucleotides in length (A, B and C). This 105 nucleotide higher order repeat has apparently been duplicated twice to yield a complex repeated sequence, ABCA'B'C'A"B"C", which exhibits divergence among the individual subunits. This sequence is AT rich, as are the direct terminal repeats which flank the copia coding region, but does not contain any apparent homology with the terminal repeats. This repeated sequence contains three presumptive polyadenylation signals and two 25 nucleotide, imperfectly matched, inverted repeat sequences adjacent to two of the polyadenylation sequences.

Animals↗