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Biomedical subjects

J W Fox

Publications and source records attributed to J W Fox.

At least 55 records · Page 3Linked to original sources
PubMed1998

Epidermal growth factor augments adaptation following small bowel resection: optimal dosage, route, and timing of administration.

BACKGROUND: In assorted animal models of small bowel resection (SBR), exogenous epidermal growth factor (EGF) has been shown to augment intestinal adaptation. This study was designed to elucidate the optimal dose, route, and timing of exogenous EGF to boost adaptation in our murine model of SBR. METHODS: Male ICR mice underwent either 50% proximal SBR or sham surgery (bowel transection with reanastomosis) and then randomized to receive either saline or human recombinant EGF (5, 50, 150, or 300 microg/kg/day) by twice daily intraperitoneal (i.p.) injection or orogastric gavage (o.g.). At 7 days, protein and DNA content, crypt depth, and villus height were determined in the ileum. The premium dose and route was then given for 1 week either during (1 week after SBR) or after the adaptive phase (1 month after SBR). Differences between group means were analyzed using ANOVA. A P < 0.05 was considered significant. RESULTS: EGF enhanced DNA and protein content, crypt depth, and villus height to the greatest extent at a dosage of 50 microg/kg/day by the o.g. route. EGF had no significant effect on enhancing adaptation when given after the adaptive response had already occurred. CONCLUSIONS: Intestinal adaptation is optimally enhanced by a specific dose and route of EGF. Exogenous EGF enhances adaptation only during the adaptive response to SBR and not after it has already taken place. Determination of the best circumstances for EGF administration will permit a systematic approach toward understanding a mechanism for the beneficial effect of EGF during intestinal adaptation.

Adaptation, Physiological↗
PubMed1998

Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia.

Long-range, directed migration is particularly dramatic in the cerebral cortex, where postmitotic neurons generated deep in the brain migrate to form layers with distinct form and function. In the X-linked dominant human disorder periventricular heterotopia (PH), many neurons fail to migrate and persist as nodules lining the ventricular surface. Females with PH present with epilepsy and other signs, including patent ductus arteriosus and coagulopathy, while hemizygous males die embryonically. We have identified the PH gene as filamin 1 (FLN1), which encodes an actin-cross-linking phosphoprotein that transduces ligand-receptor binding into actin reorganization, and which is required for locomotion of many cell types. FLN1 shows previously unrecognized, high-level expression in the developing cortex, is required for neuronal migration to the cortex, and is essential for embryogenesis.

Abnormalities, Multiple↗
PubMed1998

Adaptation after small bowel resection is attenuated by sialoadenectomy: the role for endogenous epidermal growth factor.

BACKGROUND: Epidermal growth factor (EGF) is likely involved during adaptation after small bowel resection (SBR) because some studies have shown enhanced adaptation by EGF administration. Because the major source of endogenous EGF in mice is the submandibular glands, we sought to determine the effect of submandibular gland excision (SAL) and luminal or systemic EGF replacement on adaptation after SBR. METHODS: A 50% proximal SBR or Sham-SBR (bowel transection and reanastomosis) was performed on male C57BL/6 mice after either SAL or gland mobilization only. Additional mice underwent both SBR and SAL and then received daily EGF or saline solution by intraperitoneal or orogastric administration. At 1 week, adaptation was characterized in the ileum as changes in villus height, DNA, and protein content. RESULTS: SAL significantly attenuated the increase in ileal villus height, total protein, and DNA content after SBR. Both systemic and oral EGF reversed these findings equally and significantly augmented all parameters of intestinal adaptation after SAL. CONCLUSIONS: Submandibular EGF is important for the adaptive response to massive SBR. As both luminal and systemic EGF equally reversed the findings following SAL and SBR, the specific site of action for endogenous EGF during adaptation is either the luminal or basolateral surface of the enterocyte.

Adaptation, Physiological↗
PubMed1997

Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper.

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23-25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23-25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I-IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

Amino Acid Sequence↗
PubMed1997

Sequence and biological activity of catrocollastatin-C: a disintegrin-like/cysteine-rich two-domain protein from Crotalus atrox venom.

In this study we report on the isolation and biological characterization of a 23.6-kDa protein from the venom of the western diamondback rattlesnake, Crotalus atrox. Primary structural analysis shows the protein to be composed of a spacer/disintegrin-like domain and a cysteine-rich domain. The sequence is identical to the same carboxy-terminal domains found in the C. atrox metalloproteinase, catrocollastatin, and hence we termed the protein catrocollastatin-C. We estimate that catrocollastatin-C represents at least 0.5% of the total protein in C. atrox venom. The protein is an inhibitor of collagen-stimulated but not ADP-stimulated platelet aggregation. Reduction and alkylation of catrocollastatin-C causes a loss of platelet aggregation inhibitor activity. A synthetic, cyclic peptide designed from the catrocollastatin-C disintegrin-like domain has potent platelet aggregation inhibitory activity. This suggests that the corresponding region in the disintegrin-like domain of the protein is at least partially responsible for the inhibition of platelet aggregation previously reported for the protein. These studies underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of precursor proteins and how the processed precursor fragments may contribute to the observed pathological effects of the venom.

Adenosine Diphosphate↗
PubMed1997

Function of disintegrin-like/cysteine-rich domains of atrolysin A. Inhibition of platelet aggregation by recombinant protein and peptide antagonists.

Snake venom hemorrhagic metalloproteinase toxins that have metalloproteinase, disintegrin-like and cysteine-rich domains are significantly more potent than toxins with only a metalloproteinase domain. The disintegrin-like domains of these toxins differ from the disintegrin peptides found in crotalid and viperid venoms by the nature of their different disulfide bond structure and, in lieu of the disintegrins' signature Arg-Gly-Asp (RGD) integrin binding sequence, there is an XXCD disulfide-bonded cysteinyl sequence in that region. Due to these apparent differences, the contribution to the overall function of the hemorrhagic metalloproteinases by the disintegrin-like domain has been unknown. In this investigation we have expressed in insect cells the disintegrin-like/cysteine-rich (DC) domains of the Crotalus atrox hemorrhagic metalloproteinase atrolysin A and demonstrated that the recombinant protein (A/DC) can inhibit collagen- and ADP-stimulated platelet aggregation. Using synthetic peptides, we have evidence that the region of the disintegrin-like domain that is positionally analogous to the RGD loop of the disintegrins is the site responsible for inhibition of platelet aggregation. For these synthetic peptides to have significant inhibitory activity, the -RSECD- cysteinyl residue must be constrained by participation in a disulfide bond with another cysteinyl residue. The two acidic amino acids adjacent to the middle cysteinyl residue in these peptides are also important for biological activity. These studies emphasize a functional role for the disintegrin-like domain in toxins and suggest structural possibilities for the design of antagonists of platelet aggregation.

Amino Acid Sequence↗
PubMed1997

Dexamethasone stimulates leptin release from human adipocytes: unexpected inhibition by insulin.

In the present study we have examined the effect of dexamethasone on ob gene mRNA expression and leptin release from isolated human subcutaneous adipocytes. Dexamethasone stimulated leptin release from cultured adipocytes in a time- and dose-dependent manner. A two-fold increase in leptin release was detectable by 36 h of treatment with 10(-7) M dexamethasone. Leptin release was preceded by a significant 83 +/- 30% increase in ob mRNA after 24 h exposure to the compound. Co-incubation of cells with dexamethasone (10(-7) M) and insulin (10(-7) or 10(-9) M) completely blocked the dexamethasone-stimulated increase in ob mRNA and leptin release. These data demonstrate that insulin and glucocorticoids regulate leptin synthesis and release from human adipocytes in vitro.

Adipocytes↗
PubMed1997

Isolation and characterization of a potent curarizing polypeptide from Tityus discrepans scorpion venom.

The pure TdI-1 polypeptide that blocks miniature endplate potentials (MEPPs) and abolishes or reduces endplate potentials (EPPs) below the action potential threshold was identified from the crude fraction of Tityus discrepans venom. The toxin is a potent reversible non-depolarizing muscle relaxant that blocks more than 95% of the EPP at a 2 microM (0.1 mg/ml) concentration. On a molar basis, TdI-1 is as potent as or more potent than many muscle relaxants since, at the concentration used, the toxin suppressed more than 95% of the EPP. Using matrix-assisted laser desorption time of flight (MALD-TOF) ionization mass spectrometry, TdI-1 was found to have an unusally large mol. wt for a scorpion toxin, close to 48,000. The N-terminal sequence of the first 23 residues of TdI-1 was also determined. The fragment differs from the N-terminal sequences of all 140 peptidic scorpion toxins found in the SWISSPROT and PIR databases using the search engine of the felix.EMBL-Heidelberg.de computer (European Molecular Biology Laboratory, Heidelberg, Germany.

Amino Acid Sequence↗
PubMed1997

Aberrant splicing of a mouse disabled homolog, mdab1, in the scrambler mouse.

Although accurate long-distance neuronal migration is a cardinal feature of cerebral cortical development, little is known about control of this migration. The scrambler (scm) mouse shows abnormal cortical lamination that is indistinguishable from reeler. Genetic and physical mapping of scm identified yeast artificial chromosomes containing an exon of mdab1, a homolog of Drosophila disabled, which encodes a phosphoprotein that binds nonreceptor tyrosine kinases. mdab1 transcripts showed abnormal splicing in scm homozygotes, with 1.5 kb of intracisternal A particle retrotransposon sequence inserted into the mdab1 coding region in antisense orientation, producing a mutated and truncated predicted protein. Therefore, mdab1 is most likely the scm gene, thus implicating nonreceptor tyrosine kinases in neuronal migration and lamination in developing cerebral cortex.

Animals↗
PubMed1997

Mucosal injury and disruption of intestinal barrier function in HIV-infected individuals with and without diarrhea and cryptosporidiosis in northeast Brazil.

OBJECTIVES: To determine the relative effects of AIDS-related diarrhea with or without cryptosporidiosis and microsporidiosis on intestinal function and injury. METHODS: We studied 40 HIV-infected patients (20 with and 20 without diarrhea) and 13 healthy volunteers, using the differential urinary excretion of ingested lactulose and mannitol as respective markers of barrier disruption and overall villous surface area. We also examined them for fecal leukocytes, lactoferrin, and alpha 1-antitrypsin. Fasting subjects drank test solution containing lactulose (5 g) and mannitol (1 g). Urine was collected for 5 h and tested for sugars by high-performance liquid chromatography with pulsed amperometric detection. RESULTS: HIV-positive patients with diarrhea had a 2.8-fold higher lactulose:mannitol excretion ratio (L:M) than HIV-positive patients without diarrhea (p = 0.01) and 10.4-fold higher than healthy volunteers (p = 0.004). This was accounted for by a 1.5- to 3.1-fold higher rate of lactulose excretion by HIV patients with diarrhea than by those without diarrhea or by healthy volunteers. Mannitol excretion was 32-55% less in patients with diarrhea than in those without diarrhea or in healthy volunteers. Patients with cryptosporidial diarrhea had a nearly 6-fold higher L:M ratio than those without diarrhea (p < 0.001) and nearly 3-fold higher than those with non-cryptosporidial diarrhea (p = 0.02). One patient with microsporidial infection had a nearly 3-fold higher L:M ratio than controls without diarrhea. Alpha 1-Antitrypsin was positive in 40% of HIV-positive patients with cryptosporidial infections and none of 12 HIV-positive patients with non-cryptosporidial diarrhea. Fecal lactoferrin or leukocytes were increased in all HIV patients with diarrhea. CONCLUSION: HIV infection is associated with intestinal dysfunction and injury, even in patients who do not have diarrhea. However, those with diarrhea, especially with cryptosporidiosis or microsporidiosis, have even greater disruption of intestinal barrier function with potentially important nutritional consequences.

AIDS-Related Opportunistic Infections↗
PubMed1996

Expression, activation, and processing of the recombinant snake venom metalloproteinase, pro-atrolysin E.

The expression in human embryonic kidney (HEK 293) cells of the recombinant zymogen form (pro-) of the Crotalus atrox hemorrhagic metalloproteinase, atrolysin E, is presented. The nascent protein is comprised of pre-, pro-, proteinase-, spacer-, and disintegrin domains. The biochemical characterization of the recombinant zymogen is described along with its activation by C. atrox crude venom and other hemorrhagic toxins. Unlike the zymogen forms of the matrix metalloproteinases, pro-atrolysin E is not activated by the organomercurial, (4-aminophenyl)mercuric acetate. Pro-atrolysin E could be enzymatically activated by C. atrox crude venom, PMSF-inhibited crude venom, atrolysin A, and atrolysin E itself. There is no evidence of autoactivation. Using two polyclonal antibodies directed against the proteinase domain and the disintegrin domain of atrolysin E, the proteolytic processing of the recombinant protein by atrolysin A was followed. The first cleavage of pro-atrolysin E by atrolysin A removes the pro-domain. The second proteolysis step removes the disintegrin domain to produce the proteinase/spacer protein. These studies have identified potential activators of snake venom pro-metalloproteinases in crude venom and suggest a general scheme for the activation and processing of venom pro-metalloproteinases by the endogenous, active metalloproteinases.

Amino Acid Sequence↗
PubMed1996

Direct analysis of mannitol, lactulose and glucose in urine samples by high-performance anion-exchange chromatography with pulse amperometric detection. Clinical evaluation of intestinal permeability in human immunodeficiency virus infection.

Clinically, the ratio of lactulose/mannitol excretion in urine after administration of these non-metabolized sugars has been used to evaluate the extent of malabsorption and intestinal permeability disruption in several infections and nutritional diseases, including human immunodeficiency virus (HIV) infection. A range of methodologies have been reported to determine the lactulose/mannitol ratio, including enzymatic assay, gas-liquid chromatography (GC), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Most published methods involve tedious sample preparations, rendering them unsuitable for routine or automated clinical laboratory testing. We describe in this paper a method in which weak anion-exchange high-performance liquid chromatography in conjunction with a pulsed amperometric detector was used. It requires very simple sample preparation and avoids interference by other components present in the urine. The linear range of determination for mannitol, lactulose and glucose are up to 10 nmol, in a single injection. The limits of detection are 8, 12, 47 and 52 pmol, respectively, for mannitol, glucose, lactose and lactulose. The separation and quantification using this method are highly reproducible, yielding standard errors of less than 2.5% for retention times and less than 3.5% for quantitation. The ratios of lactulose/mannitol recovery in controls and in HIV-infected subjects with and without diarrhea showed striking differences, which are in close agreement with the published results derived with similar HPLC methods.

Calibration↗
PubMed1995

Platelet-derived growth factors-AA and -BB regulate collagen and collagenase gene expression differentially in human fibroblasts.

Platelet-derived growth factor (PDGF) is a mitogen associated with tissue repair, a process involving collagen synthesis and remodelling by interstitial collagenase. This study examines and compares the regulation of interstitial collagenase and collagen gene expression by PDGF-AA and -BB in human fibroblasts. Time-course analysis showed that neither PDGF-AA or -BB had a consistent effect on the expression of pro-alpha 1(I) or pro-alpha 2(I) type-I collagen genes. In contrast, interstitial collagenase gene expression was found to be consistently up-regulated severalfold by PDGF-BB. Enhanced expression of the collagenase gene was not apparently due to up-regulation of its promoter activity in human dermal fibroblasts, as indicated by transient and stable transfection experiments. Unlike PDGF-BB, PDGF-AA did not alter collagenase mRNA levels under low-serum culture conditions. Thus, the biological activities of the PDGF homodimers are different, with PDGF-BB being clearly more potent than PDGF-AA in its regulation of collagenase gene expression.

Becaplermin↗
PubMed1995

Molecular cloning of a major cockroach (Blattella germanica) allergen, Bla g 2. Sequence homology to the aspartic proteases.

Inhalation of allergens produced by the German cockroach (Blattella germanica) elicits IgE antibody formation and the development of asthma in genetically predisposed individuals. We compared the allergenic importance of two cockroach (CR) allergens, Bla g 1 and Bla g 2, and determined the complete amino acid sequence of the major 36-kDa allergen, Bla g2. A survey of 106 sera from CR allergic patients showed the prevalence of IgE antibodies to Bla g 1 and Bla g 2 to be 30.2% and 57.6%, respectively. Immediate skin tests on 7 selected patients gave positive reactions using 10(-3) micrograms/ml either allergen, whereas controls showed no response to 10 micrograms/ml. Natural Bla g 2 was purified and the sequence of the NH2 terminus and tryptic peptides, comprising 36% of the molecule, was determined. The cDNA for Bla g 2 was cloned from a B. germanica expression library and encoded a 24-amino acid signal peptide and a 328-amino acid mature protein, which showed the highest degree of identity to mosquito (Aedes aegypti) lysosomal aspartic protease (30.8%), with similar identity to pepsin, cathepsins D and E, renin, and chymosin. Bla g 2 mRNA and protein were detected in B. germanica, but not in Periplaneta americana, the other principal domiciliary CR species in the U.S. High concentrations of Bla g 2 were found in CR digestive organs (esophagus, gut, and proventriculus). The results show that Bla g 2 is a major species-specific allergen of B. germanica and suggest that the allergen functions as a digestive enzyme in the cockroach.

Allergens↗
PubMed1995

Structure-based analysis of inhibitor binding to Ht-d.

A theoretical study was performed on the structure of both the native and inhibited metalloproteinase Ht-d (E.C. 3.4.24.42) solved at 2.0 A resolution. The energy maps calculated by program GRID clearly showed the extended binding site of Ht-d and allowed localization and characterization of the pockets S1-S3 and S1'-S3'. The GRID energy contour maps point out the particular shape of the S1' pocket in agreement with experimental density maps and inhibited Ht-d structures. Based on the high degree of sequence homology of the Ht-d active site to that of mammalian metalloproteinases, the characterization of active site pockets was extended to neutrophil collagenase, fibroblast collagenase, stromelysin 1 and 2. Thirty residues of the Ht-d propeptide were modeled and optimized with reference to the Ht-d structure, giving insight to the mechanism of natural inhibition in metalloproteinase proenzymes. Kinetic measurements of Ht-d inhibition by a series of synthetic peptides show, in agreement with our Ht-d propeptide model, the crucial role of cysteine and adjacent residues in the specificity of Ht-d propeptide. This study suggests the structural link between Ht-d and mammalian metalloproteinases, contributing to the understanding of the mechanism of natural and synthetic inhibitor binding to metalloproteinases. Therefore, Ht-d is a good model system for the design of novel inhibitors against these enzymes with enhanced potency and specificity.

Journal Article↗
PubMed1995

Primary structure of a coagulant enzyme, bilineobin, from Agkistrodon bilineatus venom.

The amino acid sequence and disulfide bridge location of the coagulant enzyme, named bilineobin, isolated from the venom of Agkistrodon bilineatus was determined by Edman sequencing of the peptides derived from digests with cyanogen bromide, clostripain, Staphylococcus aureus V8 protease, trypsin, and chymotrypsin. This enzyme has a molecular weight of 57,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, bilineobin consists of 235 amino acids and has a calculated molecular weight of 26,481. The enzyme contains fucose, GlcNAc, galactose, mannose and NeuAc and six N-linked glycosylation consensus sites. The carboxyterminal amino acid, proline, was determined using carboxypeptidase Y. The six disulfide bonds of bilineobin link Cys78 to Cys234, Cys120 to Cys188, Cys178 to Cys203, Cys7 to Cys141, Cys152 to Cys167, and Cys28 to Cys44. The amino acid sequence similarity to flavoxobin (T.C. Shieh et al., 1988, J. Biochem (Tokyo) 103, 596-605) and batroxobin (N. Itoh et al., 1987, J. Biol. Chem. 262, 3132-3135) was 67%. The deglycosylated enzyme more rapidly generated fibrinopeptide A than native bilineobin.

Agkistrodon↗