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Biomedical subjects

G Gimenez-Gallego

Publications and source records attributed to G Gimenez-Gallego.

At least 19 recordsLinked to original sources
PubMed1992

Fibroblast growth factor injected in cerebral ventricles does not decrease mean arterial blood pressure.

Intracerebroventricular injections of acidic fibroblast growth factor (aFGF) in rats did not elicit any change in the mean arterial blood pressure, and also did not appreciably affect the hypotensive effect of intravenous injections of aFGF. These observations are of general clinical interest, since such effects could constitute important drawbacks for the therapeutic applications of FGF.

Animals↗
PubMed1992

Localization of acidic fibroblast growth factor within the mouse brain using biochemical and immunocytochemical techniques.

The localization of acidic fibroblast growth factor (aFGF) in the male mouse brain was studied with biochemical and immunocytochemical techniques. Using two peptide-based aFGF antisera directed against independent epitopes, Western gel analysis of dissected brain demonstrated significant levels of aFGF immunoreactivity in the pons-medulla, hypothalamus and cerebellum. The cortex contained much less immunoreactivity. Consistent with the biochemical data, immunocytochemical analysis with the same two antisera demonstrated that aFGF immunoreactivity is localized in neuronal cell bodies in these regions. Numerous immunoreactive neurons were observed in the reticular formation of the pons and medulla, as well as in several other brainstem nuclei and areas. Immunoreactive neurons were also present in the lateral and medial hypothalamus, and some thalamic, subthalamic and epithalamic nuclei. In the basal ganglia, immunoreactive neurons were present in the amygdala and septum. Few intensely stained immunoreactive neurons were observed in the striatum, pallidum and neocortex. Limbic cortices contained more numerous immunoreactive neurons than neocortex. These results support the concept that aFGF is present in the brain, where it is heterogeneously distributed in neuronal cell bodies in regions involved in sensory, extrapyramidal motor, limbic and autonomic functions. The results are consistent with various neurotrophic, mitogenic, and neuromodulatory functions associated with aFGF in the mammalian central nervous system.

Amino Acid Sequence↗
PubMed1992

Acidic fibroblast growth factor accelerates dermal wound healing.

Acidic fibroblast growth factor (aFGF) is a potent mitogen in vitro for many cells of ectodermal and mesodermal embryonic origin including skin-derived epidermal keratinocytes, dermal fibroblasts and vascular endothelial cells. Based on the mitogenic activity for these skin-derived cells, we tested the ability of topically applied aFGF to promote healing of full-thickness dermal wounds in healthy rodents. Low doses of aFGF can produce almost a two-fold maximum acceleration in the rate of closure of full-thickness dermal punch biopsy wounds in young healthy mice and rats. The mitogen also produces a 3 to 4 day acceleration in the time to complete closure in rats. Quantitative histomorphometric analysis of wound tissue shows that aFGF induces a marked stimulation of angiogenesis, granulation tissue formation and the growth of new epithelium, but does not promote dermal contraction. Application of aFGF to linear incisions in rat skin produces a transient increase in wound tensile strength accompanied by enhanced cellularity and deposition of collagen. Therefore, aFGF functions as a pharmacological agent that can accelerate dermal wound healing in rodents and could act therapeutically to promote dermal tissue repair in humans.

3T3 Cells↗
PubMed1991

Conversion of cysteine to serine residues alters the activity, stability, and heparin dependence of acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is a broad spectrum mitogen that is stabilized by complexation with heparin and heparan proteoglycans. The monomeric human protein contains 3 reduced cysteine residues of unknown function, the first 2 of which are conserved among all seven known fibroblast growth factors. The influence of these free sulfhydryl groups on the level, stability, and heparin dependence of the mitogenic activity at physiological temperature and pH is characterized using a complete set of site-directed mutants in which either any 1, 2, or all 3 of the cysteine residues are converted to serines. Mutants of aFGF in which either any 2 or all 3 cysteine residues are substituted by serines are more active, have longer activity half-lives, and are less heparin dependent than wild-type aFGF. In contrast, wild-type aFGF and the three mutants that each retain 2 cysteine residues inactivate more rapidly in the absence of heparin by a nonproteolytic mechanism but are markedly stabilized by heparin. This cysteine-mediated destabilization of aFGF not only diminishes its activity in the absence of heparin in tissue culture but also could functionally restrict its activity in vivo to the vicinity of mast cell-derived heparins and heparan proteoglycans associated with cell surfaces and basement membranes.

Blotting, Western↗
PubMed1991

Immunohistochemical localization of basic fibroblast growth factor in ependymal cells of the rat lateral and third ventricles.

The presence of basic fibroblast growth factor (bFGF) in the third and the lateral ventricular ependyma of the rat has been investigated under the light microscope using a polyclonal antibody against bFGF, through the unlabelled peroxidase-antiperoxidase procedure; bFGF-immunoreactivity (bFGF-IR) is observed in the entire ependymal cell layer of ventricles. Also ependymal tanycytes within the inferior portion of the wall and floor of the third ventricle show bFGF-IR. Tanycytic processes are in close contact with hypothalamic capillaries. The present study strongly suggests that brain ependymal bFGF plays unidentified roles unrelated to its angiogenic or mitogenic capacities.

Animals↗
PubMed1990

Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus.

An inhibitor of the high conductance, Ca2(+)-activated K+ channel (PK,Ca) has been purified to homogeneity from venom of the scorpion Buthus tamulus by a combination of ion exchange and reversed-phase chromatography. This peptide, which has been named iberiotoxin (IbTX), is one of two minor components of the crude venom which blocks PK,Ca. IbTX consists of a single 4.3-kDa polypeptide chain, as determined by polyacrylamide gel electrophoresis, analysis of amino acid composition, and Edman degradation. Its complete amino acid sequence has been defined. IbTX displays 68% sequence homology with charybdotoxin (ChTX), another scorpion-derived peptidyl inhibitor of PK,Ca, and, like this latter toxin, its amino terminus contains a pyroglutamic acid residue. However, IbTX possesses 4 more acidic and 1 less basic amino acid residue than does ChTX, making this toxin much less positively charged than the other peptide. In single channel recordings, IbTX reversibly blocks PK,Ca in excised membrane patches from bovine aortic smooth muscle. It acts exclusively at the outer face of the channel and functions with an IC50 of about 250 pM. Block of channel activity appears distinct from that of ChTX since IbTX decreases both the probability of channel opening as well as the channel mean open time. IbTX is a selective inhibitor of PK,Ca; it does not block other types of voltage-dependent ion channels, especially other types of K+ channels that are sensitive to inhibition by ChTX. IbTX is a partial inhibitor of 125I-ChTX binding in bovine aortic sarcolemmal membrane vesicles (Ki = 250 pM). The maximal extent of inhibition that occurs is modulated by K+, decreasing as K+ concentration is raised, but K+ does not affect the absolute inhibitory potency of IbTX. A Scatchard analysis indicates that IbTX functions as a noncompetitive inhibitor of ChTX binding. Taken together, these data suggest that IbTX interacts at a distinct site on the channel and modulates ChTX binding by an allosteric mechanism. Therefore, IbTX defines a new class of peptidyl inhibitor of PK,Ca with unique properties that make it useful for investigating the characteristics of this channel in target tissues.

Amino Acid Sequence↗
PubMed1990

Disulfide bonds are neither required, present, nor compatible with full activity of human recombinant acidic fibroblast growth factor.

Human acidic fibroblast growth factor (aFGF) is a potent broad-spectrum mitogen that contains three Cys residues within its monomeric structure. We have found that site-directed mutants in which any one of these Cys residues is converted to serine remain highly active, although variably dependent on heparin, so none of the three possible intramolecular disulfide bonds that can be formed are required for mitogenic activity. Furthermore, a dispensable disulfide bond that might stabilize the active conformation is not present since all three Cys residues are accessible to chemical modification in recombinant as well as brain-derived aFGFs. Finally, formation of a disulfide bond between the two Cys residues conserved among all seven known members of the FGF family results in a virtually inactive product that can subsequently be reactivated by reduction. Thus, despite the extracellular function of aFGF, its Cys residues do not form intramolecular disulfide bonds in the active conformation.

Animals↗
PubMed1988

Purification and characterization of a unique, potent inhibitor of apamin binding from Leiurus quinquestriatus hebraeus venom.

An inhibitor of apamin binding has been purified to homogeneity in three chromatographic steps from the venom of the scorpion, Leiurus quinquestriatus hebraeus. The inhibitor, which we have named leiurotoxin I, represents less than 0.02% of the venom protein. It is a 3.4-kDa peptide with little structural homology to apamin although it has some homology to other scorpion toxins such as charybdotoxin, noxiustoxin, and neurotoxin P2. Leiurotoxin I completely inhibits 125I-apamin binding to rat brain synaptosomal membranes (Ki = 75 pM). Thus, it is 10-20-fold less potent than apamin. Leiurotoxin I is not a strictly competitive inhibitor of this binding reaction. Like apamin, leiurotoxin I blocks the epinephrine-induced relaxation of guinea pig teniae coli (ED50 = 6.5 nM), while having no effect on the rate or force of contraction in guinea pig atria or rabbit portal vein preparations. Thus, leiurotoxin I of scorpion venom and apamin of honeybee venom demonstrate similar activities in a variety of tissues, yet are structurally unrelated peptides. These two peptides should be useful in elucidating the role of the small conductance, Ca2+-activated K+ channels in different tissues.

Amino Acid Sequence↗
PubMed1988

Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels.

Charybdotoxin (ChTX), a protein present in the venom of the scorpion Leiurus quinquestriatus var. hebraeus, has been purified to homogeneity by a combination of ion-exchange and reversed-phase chromatography. Polyacrylamide gel electrophoresis, amino acid analysis, and complete amino acid sequence determination of the pure protein reveal that it consists of a single polypeptide chain of 4.3 kDa. Purified ChTX is a potent and selective inhibitor of the approximately 220-pS Ca2+-activated K+ channel present in GH3 anterior pituitary cells and primary bovine aortic smooth muscle cells. The toxin reversibly blocks channel activity by interacting at the external pore of the channel protein with an apparent Kd of 2.1 nM. The primary structure of ChTX is similar to a number of neurotoxins of diverse origin, which suggests that ChTX is a member of a superfamily of proteins that modify ion-channel activities. On the basis of this similarity, the three-dimensional structure of ChTX has been modeled from the known crystal structure of alpha-bungarotoxin. These studies indicate that ChTX is useful as a probe of Ca2+-activated K+-channel function and suggest that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block.

Amino Acid Sequence↗
PubMed1987

High-performance liquid chromatography of phenylthiocarbamyl-amino acids. Application to carboxyl-terminal sequencing of proteins.

A carboxyl-terminus microsequencing method has been developed on the basis of carboxypeptidase A digestion and derivatization of released amino acids with phenylisocyanate followed by subsequent high-performance liquid chromatographic analysis of the resulting phenylthiocarbamyl amino acids. This technique has been used to confirm the carboxyl-terminal sequence of bovine brain-derived acidic fibroblast growth factor. The method is of general application for the microsequencing of the carboxyl-termini of proteins. Since identification of derivatized amino acids was performed on the entire reaction mixture following digestion, a new chromatographic identification procedure for phenylthiocarbamyl amino acids was developed in order to resolve them from spurious peaks. This procedure can be applicable to amino acid analysis.

Amino Acids↗
PubMed1986

The complete amino acid sequence of human brain-derived acidic fibroblast growth factor.

Acidic fibroblast growth factor is a potent mitogen for a variety of cells in culture, including vascular endothelial cells, and is angiogenic in vivo. The complete amino acid sequence of human brain-derived acidic fibroblast growth factor has been determined from amino terminal sequence analysis and carboxypeptidase A digestion of the whole protein and sequence analyses of peptides generated by tryptic, Staphylococcus aureus V8 protease and cyanogen bromide cleavages. A potential Asn-Gly-Ser glycosylation sequence is present in the human protein. The complete amino acid sequence is compared to that of the equivalent protein purified from bovine brain.

Amino Acid Sequence↗
PubMed1986

Human brain-derived acidic and basic fibroblast growth factors: amino terminal sequences and specific mitogenic activities.

Extended amino terminal sequence determinations, made on both acidic and basic fibroblast growth factors from human brain, showed extensive homology with each other and with their respective bovine counterparts. Both human growth factors in the presence of heparin have equivalent specific mitogenic activities on human umbilical vein endothelial cells in culture whereas in the absence of heparin, the acidic mitogen is less than 1% as active as the basic growth factor.

Amino Acid Sequence↗
PubMed1985

Brain-derived acidic fibroblast growth factor: complete amino acid sequence and homologies.

Bovine brain-derived acidic fibroblast growth factor (aFGF) is a protein mitogen originally identified in partially purified preparations of whole brain. The protein was purified to homogeneity and shown to be a potent vascular endothelial cell mitogen in culture and angiogenic substance in vivo. The homology of aFGF to human interleukin-1 beta was inferred from partial sequence data. The complete amino acid sequence of aFGF has now been determined and observed to be similar to both basic FGF and interleukin-1's. A neuropeptide-like sequence, flanked by basic dipeptides, was observed within the aFGF sequence.

Amino Acid Sequence↗
PubMed1985

A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II.

Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross-reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF-II belong to a common family of polypeptide growth factors.

Animals↗
PubMed1982

Photooxidase system of Rhodospirillum rubrum III. The role of rhodoquinone and ubiquinone in the activity of preparations of chromatophores and photoreaction centers.

The role of rhodoquinone and ubiquinone in the oxygen photoreducing (photooxidase) activity of Rhodospirillum rubrum was investigated. The sole addition of purified rhodoquinone restored photooxidase activity in isolated chromatophores which had been extracted with organic solvents and which were apparently free of secondary acceptor ubiquinone. Rhodoquinone also enhanced photooxidase activity in photoreaction center preparations from which secondary ubiquinone seemed to have been removed. Those results suggest that rhodoquinone accepts electrons directly from primary ubiquinone during chromatophore photooxidase activity. In contrast, rhodoquinone does not participate in the basal activity of photoreaction center preparations, which seems to result from the autooxidation of both primary and secondary ubiquinone.

Bacterial Chromatophores↗
PubMed1981

Effect of ammonium ions on the aerobic glycolysis in Ehrlich ascites tumor cells.

The effect of ammonium ions on aerobic glycolysis has been studied in Ehrlich ascites tumor cells. The inhibition of respiration caused by 5-25 mM glucose, fructose or mannose is increased by the presence of 0.5-20 mM NH4+. Under these conditions, more glucose is used and a crossover analysis of intermediate metabolites of glycolysis indicates an increase in the glycolytic flux. In spite of the stimulation of glycolysis, the production of lactate decreases in the presence of ammonium ions, although the concentration of free alanine (glutamate and aspartate) is increased. Ammonium ions enhance the oxidation of cytochromes c and a caused by glucose, and decrease the adenine nucleotide pool and the ATP/ADP ratio. These effects of ammonium ions are observed when glucose is present in the incubation medium, and do not occur when only ammonium salts were supplied to the cells metabolizing endogenous substrates.

Adenine Nucleotides↗