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Biomedical subjects

J W Fox

Publications and source records attributed to J W Fox.

At least 37 records · Page 2Linked to original sources
PubMed2000

Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties.

Myotoxic phospholipases A(2) (PLA(2)s; group II) account for most of the muscle-tissue damage that results from envenomation by viperid snakes. In the venom of the Godman's viper (Cerrophidion godmani, formerly Bothrops godmani), an enzymically active PLA(2) (myotoxin I) and an inactive, Lys-49 variant (myotoxin II) induce extensive muscle damage and oedema. In this study, two distinct myotoxin inhibitor proteins of C. godmani, CgMIP-I and CgMIP-II, were purified directly from blood plasma by selective binding to affinity columns containing either myotoxin I or myotoxin II, respectively. Both proteins are glycosylated, acidic (pI=4) and composed of 20-25-kDa subunits that form oligomers of 110 kDa (CgMIP-I) or 180 kDa (CgMIP-II). In inhibition studies, CgMIP-I specifically neutralized the PLA(2) and the myotoxic, oedema-forming and cytolytic activities of myotoxins I, whereas CgMIP-II selectively inhibited the toxic properties of myotoxin II. N-terminal amino acid sequence analysis and sequencing of cDNAs encoding the two inhibitors revealed that CgMIP-I is similar to gamma-type inhibitors, which share a pattern of cysteine residues present in the Ly-6 superfamily of proteins, whereas CgMIP-II shares sequence identity with alpha-type inhibitors that contain carbohydrate-recognition-like domains, also found in C-type lectins and mammalian PLA(2) receptors. N-terminal sequencing of myotoxin I revealed a different primary structure from myotoxin II [De Sousa, Morhy, Arni, Ward, Díaz and Gutiérrez (1998) Biochim. Biophys. Acta 1384, 204-208], which provides insight into the nature of such pharmacological specificity.

Amino Acid Sequence↗
PubMed2000

Inhibition of platelet aggregation by the recombinant cysteine-rich domain of the hemorrhagic snake venom metalloproteinase, atrolysin A.

The P-III class of venom metalloproteinases has, in addition to the proteinase domain, a disintegrin-like domain and a cysteine-rich domain. Recent evidence has shown that the nonproteinase domains of the P-III class of hemorrhagic metalloproteinases function in the inhibition of platelet aggregation by blocking essential procoagulant integrins on platelets. A specific role for the highly conserved cysteine-rich domain has yet to be described. In this study, we expressed the cysteine-rich domain from the hemorrhagic metalloproteinase atrolysin A and demonstrated its ability to inhibit collagen-stimulated platelet aggregation. Additionally, the cysteine-rich domain was shown to interact with MG-63 cells to inhibit adhesion to collagen I. These data suggest a functional role for the cysteine-rich domain of the P-III toxins in the observed coagulopathy by targeting the toxin to platelets and inhibiting collagen-stimulated platelet aggregation. These characteristics may function to synergistically increase the hemorrhagic effect of the toxins.

Amino Acid Sequence↗
PubMed2000

Periventricular nodular heterotopia in patients with filamin-1 gene mutations: neuroimaging findings.

BACKGROUND: The filamin-1 (FLN-1) gene is responsible for periventricular nodular heterotopia (PNH), which is an X-linked dominant neuronal migration disorder. OBJECTIVE: To review the clinical and imaging findings in a series of patients with documented filamin-1 mutations. MATERIALS AND METHODS: A retrospective review of the medical records and MR studies of a series of patients with PNH and confirmed FLN-1 mutations was done. There were 16 female patients (age range: .67-71 years; mean = 28.6) with filamin-1 gene mutations. RESULTS: In six of the patients the same mutation was inherited in four generations in one pedigree. In a second pedigree, a distinct mutation was found in two patients in two generations. In a third pedigree, a third mutation was found in four patients in two generations. The remaining four patients had sporadic de novo mutations that were not present in the parents. Ten patients had seizures, and all patients had normal intelligence. In all 16 patients MR demonstrated bilateral near-continuous PNH. There were no consistent radiographic or clinical differences between patients carrying different mutations. CONCLUSION: Patients with confirmed FLN-1 gene mutations are usually female and have a distinctive MR pattern of PNH. Other female patients with this same MR pattern probably harbor FLN-1 mutations and risk transmission to their progeny. This information is important for genetic counseling.

Adolescent↗
PubMed2000

Mass spectrophotometric evidence for P-III/P-IV metalloproteinases in the venom of the Boomslang (Dispholidus typus).

The Boomslang, Dispholidus typus, is a mid- to rear-fanged arboreal colubrid widely distributed throughout much of the African continent. Envenoming by this species is rare although deaths have been recorded. Typical symptoms associated with envenoming include diffuse intravascular coagulation (DIC) caused by fibrinogen consumption and consequent incoagulable blood together with haemorrhage into tissues such as muscle and brain; together, these procoagulant and haemorrhagic effects of the venom result in a very poor prognosis in patients who receive a large dose of venom and who are not treated with antivenom. Renal failure may also result from acute tubular necrosis resulting from pigment nephropathy. Little is known about the toxic components present in the venom; however, proteolytic activity has been reported although the proteinases involved have not been identified. In this study we provide LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) data supporting the presence of class P-III/P-IV snake venom metalloproteinases (SVMPs) in Boomslang venom. Using a polyclonal antibody raised against the P-III haemorrhagic toxin (Jararhagin) obtained from the venom of the Brazilian pit viper, Bothrops jararaca, we identified by western blot a 65 kDa protein from Boomslang venom which cross-reacted with the jararhagin antibody. A corresponding band from SDS-PAGE was subjected to tryptic digestion followed by LC/MS/MS sequence analysis of the digestion mixture. A variety of peptide sequences were identified in the digest, one of which was clearly homologous with a highly conserved region of the disintegrin-like domains of P-III/P-IV SVMPs. These data provide the first structural evidence for the presence of SVMPs in Boomslang venom; it is possible that SVMPs may also be present in the venoms of other colubrids, which cause similar symptoms in envenomed humans. In other snake venoms, most notably those of the Viperinae and Crotalinae subfamilies, many of the coagulopathic and haemorrhagic syndromes associated with systemic and local envenoming are attributed to SVMPs. The identification of a P-III/P-IV SVMP sequence in D. typus venom suggests that many of the pathological signs resulting from envenoming by this species may also be due to the presence of SVMPs in the venom. It is hoped that these results may accelerate research into colubrid venoms and may provide new insights into novel and more efficacious treatments for colubrid envenoming.

Amino Acid Sequence↗
PubMed2000

The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom.

The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.

Amino Acid Motifs↗
PubMed1999

Differential localization of 5- and 15-lipoxygenases to the nuclear envelope in RAW macrophages.

Leukotriene formation is initiated in myeloid cells by an increase in intracellular calcium and translocation of 5-lipoxygenase from the cytoplasm to the nuclear envelope where it can utilize arachidonic acid. Monocyte- macrophages and eosinophils also express 15-lipoxygenase, which converts arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid. Enhanced green fluorescent 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) fusion proteins were expressed in the cytoplasm of RAW 264.7 macrophages. Only 5-lipoxygenase translocated to the nuclear envelope after cell stimulation, suggesting that differential subcellular compartmentalization can regulate the generation of leukotrienes versus 15(S)-hydroxyeicosatetraenoic acid in cells that possess both lipoxygenases. A series of truncation mutants of 5-LO were created to identify putative targeting domains; none of these mutants localized to the nuclear envelope. The lack of targeting of 15-LO was then exploited to search for specific targeting motifs in 5-LO, by creating 5-LO/15-LO chimeric molecules. The only chimera that could sustain nuclear envelope translocation was one which involved replacement of the N-terminal 237 amino acids with the corresponding segment of 15-LO. Significantly, no discrete targeting domain could be identified in 5-LO, suggesting that sequences throughout the molecule are required for nuclear envelope localization.

Amino Acid Sequence↗
PubMed1999

Insulin replacement therapy in diabetic rats using an osmotic pump normalizes expression of enzymes key to hepatic carbohydrate metabolism.

Intensively treating type I diabetics with continuous subcutaneous insulin infusions or multiple daily insulin injections to normalize mean blood glucose concentrations significantly reduces the onset of secondary diabetic complications when compared to conventionally treated diabetics. Our studies focused on characterizing hepatic enzyme expression in intensively and conventionally treated diabetic rats. Alloxan-induced diabetic rats were conventionally treated with insulin injected twice daily or intensively treated with similar daily dosages of insulin administered via a surgically implanted osmotic pump. Our results demonstrate a significant difference in hepatic enzyme expression when these treatment regimes are compared. In conventionally treated diabetic rats, phosphoenolpyruvate carboxykinase (PEPCK) protein and mRNA levels remained slightly elevated when compared to normal animals, glycogen phosphorylase (GP) protein levels were still slightly decreased, and glycogen synthase (GS) protein and mRNA levels remained at the elevated levels observed in untreated diabetics. In contrast, the protein and mRNA levels of all three enzymes were normalized in the insulin pump-treated animals. These results suggest that intensive insulin therapy improves glycemia directly by normalizing hepatic gene expression while conventional insulin therapy normalizes plasma glucose concentrations indirectly.

Alloxan↗
PubMed1999

Expression of the CYP4F3 gene. tissue-specific splicing and alternative promoters generate high and low K(m) forms of leukotriene B(4) omega-hydroxylase.

Cytochrome P450 4F3 (CYP4F3) catalyzes the inactivation of leukotriene B(4) by omega-oxidation in human neutrophils. To understand the regulation of CYP4F3 expression, we analyzed the CYP4F3 gene and cloned a novel isoform (CYP4F3B) that is expressed in fetal and adult liver, but not in neutrophils. The CYP4F3 gene contains 14 exons and 13 introns. The cDNAs for CYP4F3A (the neutrophil isoform) and CYP4F3B have identical coding regions, except that they contain exons 4 and 3, respectively. Both exons code for amino acids 66-114 but share only 27% identity. When expressed in COS-7 cells, the K(m) of CYP4F3B was determined to be 26-fold higher than the K(m) of CYP4F3A using leukotriene B(4) as a substrate. 5'-Rapid amplification of cDNA end studies reveal that the CYP4F3A and CYP4F3B transcripts have 5'-termini derived from different parts of the gene and are initiated from distinct transcription start sites located 519 and 71 base pairs (bp), respectively, from the ATG initiation codon. A consensus TATA box is located 27 bp upstream of the CYP4F3B transcription start site, and a TATA box-like sequence is located 23 bp upstream of the CYP4F3A transcription start site. The data indicate that the tissue-specific expression of functionally distinct CYP4F3 isoforms is regulated by alternative promoter usage and mutually exclusive exon splicing.

Adult↗
PubMed1999

Characterization of mutations in the gene doublecortin in patients with double cortex syndrome.

Mutations in the X-linked gene doublecortin, which encodes a protein with no dear structural homologues, are found in pedigrees in which affected females show "double cortex" syndrome (DC; also known as subcortical band heterotopia or laminar heterotopia) and affected males show X-linked lissencephaly. Mutations in doublecortin also cause sporadic DC in females. To determine the incidence of doublecortin mutations in DC, we investigated a cohort of eight pedigrees and 47 sporadic patients with DC for mutations in the doublecortin open reading frame as assessed by single-stranded conformational polymorphism analysis. Mutations were identified in each of the eight DC pedigrees (100%), and in 18 of the 47 sporadic DC patients (38%). Identified mutations were of two types, protein truncation mutations and single amino acid substitution mutations. However, pedigrees with DC displayed almost exclusively single amino acid substitution mutations, suggesting that patients with these mutations may have less of a reproductive disadvantage versus those patients with protein truncation mutations. Single amino acid substitution mutations were tightly clustered in two regions of the open reading frame, suggesting that these two regions are critical for the function of the Doublecortin protein.

Brain Diseases↗
PubMed1999

Cell adhesion to a population of laminin isoforms isolated from normal renal tissue.

To assess whether cells react differently towards a population of several laminin isoforms, as found in vivo, vs. a single isoform, we have compared the biological activity of kidney laminins to that of pure laminin 1. The kidney laminin preparation contained laminin 1 and further isoforms. Both substrates induced adhesion of a large spectrum of cell types, with kidney laminins being the most active. Unfolding of the coil-coiled conformation of the kidney isoforms negatively affected cell adhesion-promoting activity, which indicated that conformation-dependent cell binding is a characteristic feature of many or all laminins. Cellular interactions with kidney laminins were mediated by alpha3beta1 and alpha6beta1 integrins, with the contribution of alpha3beta1 being apparently lower than that of alpha6beta1 integrins. Immunofluorescence staining of vinculin and integrin subunits decorated focal adhesions on kidney laminins which differed in morphology from those formed on laminin 1 alone, in spite of the presence of the latter in the kidney preparation. These observations collectively indicate that tissue specific but often overlapping expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.

Amino Acid Sequence↗
PubMed1999

Design strategies and performance of custom DNA sequencing primers.

This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp. We provided a 300-bp test sequence and asked participants to submit sequences of 1-3 optimal sequencing primers. Submitted primers ranged from 17-24 nt and largely conformed to the preferred parameters. Submitted primers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "manually", more than by any software package. Each of 69 submitted and 95 control primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators). Approximately half of the control primers were also tested using rhodamine dye terminator reagents ("old" rhodamine dye terminators). The results indicated that primer physico-chemical characteristics thought to have a strong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G + C or Tm, strong secondary priming scores or long 3' homopolymeric stretches yielded excellent sequences with the dRhodamine dye terminator reagents, although these characteristics had a stronger effect when the old rhodamine reagents were used. The old rhodamine reagents gave sequences with a similar average read length, but the number of errors and ambiguities or "N's" was consistently higher. Moreover, the effects of the primer physico-chemical characteristics were also more evident with the old rhodamine dyes. We conclude that under optimal sequencing conditions with highly pure template and primer, many of the commonly applied primer design parameters are dispensable, particularly when using one of the new generation of sequencing reagents such as the dichlororhodamine dye terminators.

Autoanalysis↗
PubMed1999

Partial amino acid sequence and biological characterization of elegatoxin, hemorrhagic toxin from Trimeresurus elegans (Sakishimahabu) venom.

A hemorrhagic toxin, designated Elegatoxin, was isolated from the venom of Trimeresurus elegans using HW-55, DEAE-Sephacel, CM-Cellulose and Mono S column chromatographies. The purified toxin was shown to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, isoelectric electrophoresis, and Ouchterlony immunodiffusion. Elegatoxin has a molecular weight of 26,000 with an isoelectric point of 8.6. The toxin demonstrated both hemorrhagic and proteolytic activities. Hemorrhagic activity was inhibited by ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(2-amino-ethylether)N,N'-tetraacetic acid (EGTA), o-phenanthroline, and N-bromosuccinimide, but not by amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF). The minimum hemorrhagic dose was found to be 0.8 microgram/mouse. Elegatoxin possesses proteolytic activity as evidenced by hydrolyzing type IV collagen, actin and the A alpha, B beta, and gamma chains of bovine fibrinogen. This purified toxin contains 1 mol of zinc and 2 mols of calcium per mol of protein and a partial amino acid sequence was determined. The pathological and biochemical properties of Elegatoxin were investigated, and these results are reported in this paper.

Amino Acid Sequence↗
PubMed1998

Isolation, sequence analysis, and biological activity of atrolysin E/D, the non-RGD disintegrin domain from Crotalus atrox venom.

Crotalid snake venom metalloproteinases often have associated with them nonproteinase domains that may be processed from the mature proteinases. Nascent atrolysin E, from the western diamondback rattlesnake, Crotalus atrox, has a metalloproteinasedomain and a non-RGD disintegrin domain that is lacking in the mature metalloproteinase. In this studywe report on the isolation, sequence analysis, andbiological activity of the 7.4-kDa atrolysin E disintegrin domain (atrolysin E/D). Atrolysin E/D represents approximately 0.2% of the total protein fromthe crude venom. The protein begins with a glycinyl residue found in the latter part of the spacer region. The sequence of atrolysin E/D is identical to thatof the non-RGD disintegrin domain of atrolysin E. The structure is termed a non-RGD disintegrin sincein lieu of the characteristic RGD sequence, a Met-Val-Asp (MVD) is found instead. Nevertheless, the protein is a potent inhibitor of both collagen- and ADP-stimulated platelet aggregation with IC50 values of 4 and 8 nM, respectively. A cyclized synthetic peptide, Ac-CRVSMVDRNDDTC-NH2, which represents the sequence of the atrolysin E/D non-RGD loop, was demonstrated to be an effective inhibitor of platelet aggregation. Therefore, this region of atrolysin E/D's structure, as in the disintegrins proper, is important for the biological activity of the protein. Thus, like the non-RGD disintegrin barbourin from Sistrurus miliarius barbouri, a RGD sequence in the context of the disintegrin protein backbone is not an absolute requirement for platelet aggregation inhibitory activity. These data underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of the precursor metalloproteinases and exemplify how the processed fragments may contribute to the observed pathological effects of the venom.

Adenosine Diphosphate↗
PubMed1998

The interglobular domain of cartilage aggrecan is cleaved by hemorrhagic metalloproteinase HT-d (atrolysin C) at the matrix metalloproteinase and aggrecanase sites.

Two primary cleavage sites have been identified within the interglobular domain of the cartilage aggrecan core protein: one is between amino acid residues Asn 341 and Phe342, where many matrix metalloproteinases (MMP) have been shown to cleave; and the other is between amino acid residues Glu373 and Ala374. Although cleavage at the Glu373-Ala374 site is believed to play a critical role in cartilage aggrecan degradation in arthritic diseases, the enzyme responsible for cleavage at this site, "aggrecanase," has not been identified. Members of the ADAM (a disintegrin and metalloproteinase) family of proteins, which shows structural homology to the snake venom hemorrhagic metalloproteinases (reprolysins), have recently been demonstrated to be expressed in articular chondrocytes. Because many ADAM family members have a putative proteinase function, this raises the possibility that aggrecanase may be a member of this family of proteases. To examine whether reprolysins have the ability to cleave aggrecan at either the aggrecanase site or the MMP site, the snake venom hemorrhagic toxin metalloproteinase HT-d (atrolysin C) was tested for its ability to cleave bovine aggrecan monomer. Cleavage was monitored using the BC-3 antibody, which recognizes aggrecan fragments with the new NH2 terminus ARGSV generated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2 terminus FFGVG generated by cleavage at the MMP site. Cleavage at both the aggrecanase and MMP sites occurred in a concentration-dependent manner with 100 nM atrolysin C or greater. AF-28-reactive fragments were generated by 30 min of incubation, and levels were maximal by 8 h; BC-3-reactive fragments were detected at 2 h and continued to increase through 48 h, thus suggesting that atrolysin C can cleave at the MMP and aggrecanase sites. NH2-terminal aggrecan fragments generated by cleavage at the aggrecanase site were also detected using antisera recognizing the new COOH terminus, NITEGE, formed by cleavage at the Glu373-Ala374 bond, indicating that cleavage at this site does not require prior cleavage at the MMP site. These data provide the first demonstration that a reprolysin can cleave the core protein of aggrecan and the first example of a specific protease that can cleave at the aggrecanase site independent of cleavage at the MMP cleavage site.

Aggrecans↗
PubMed1998

Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein.

X-linked lissencephaly and "double cortex" are allelic human disorders mapping to Xq22.3-Xq23 associated with arrest of migrating cerebral cortical neurons. We identified a novel 10 kb brain-specific cDNA interrupted by a balanced translocation in an XLIS patient that encodes a novel 40 kDa predicted protein named Doublecortin. Four double cortex/X-linked lissencephaly families and three sporadic double cortex patients show independent doublecortin mutations, at least one of them a de novo mutation. Doublecortin contains a consensus Abl phosphorylation site and other sites of potential phosphorylation. Although Doublecortin does not contain a kinase domain, it is homologous to the amino terminus of a predicted kinase protein, indicating a likely role in signal transduction. Doublecortin, along with the newly characterized mDab1, may define an Abl-dependent pathway regulating neuronal migration.

Amino Acid Sequence↗