Biomedical subjects

J E Fox

Publications and source records attributed to J E Fox.

At least 37 records · Page 2Linked to original sources

PubMed1998

Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells.

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.

Amino Acid Sequence↗

PubMed1998

Frequency and synchrony of tetanically-induced, gamma-frequency population discharges in the rat hippocampal slice: the effect of diazepam and propofol.

Fast, rhythmic discharges, at gamma frequencies, have been evoked in the CA1 region of the hippocampus by tetanic stimulation applied via pairs of electrodes placed close to the pyramidal layer. The discharges had a latency of 65-225 ms and a frequency of 60-100 Hz; they were synchronous (phase lag < 2 ms) when recorded from sites 0.4-0.8 mm apart. Diazepam (5-15 microM) and propofol (5 microM) reduced the frequency by 15-26%, but had no significant effect on synchrony or phase lag, suggesting that the generation and synchrony of this rhythm are pharmacologically distinct. 10 microM propofol had a more marked effect and, when frequency fell by approximately 50%, synchrony was also reduced.

Animals↗

PubMed1998

Role of actin-binding protein in insertion of adhesion receptors into the membrane.

The goal of this study was to determine whether actin-binding protein (ABP) regulates membrane composition. ABP-deficient and ABP-containing cells were transfected with the cDNAs coding for glycoprotein (GP) Ib-IX, a platelet receptor that interacts with ABP. Most of the GP Ib-IX remained inside the ABP-deficient cells. When ABP was present, functional GP Ib-IX was inserted into the membrane. GP Ib-IX lacking the domain that interacts with ABP also showed increased membrane insertion in ABP-expressing cells. Furthermore, a fragment of ABP that lacks the dimerization and GP Ib-IX-binding sites restored the spreading of the cells and increased the amount of GP Ib-IX in the membrane. Finally, expression of ABP also increased endogenous beta1 integrin in the membrane. These results indicate that 1) ABP maintains the properties of the cell such that adhesion receptors can be efficiently expressed in the membrane; 2) increased receptor expression is accompanied by increased ability of the cell to spread; and 3) ABP exerts its effect by a mechanism that does not appear to involve direct cross-linking of actin filaments or direct interaction with receptors.

Cell Membrane↗

PubMed1998

Prenatal diagnosis of low level trisomy 15 mosaicism: review of the literature.

Low level chromosome mosaicism found at amniocentesis is problematic for clinicians and patients. We report prenatal diagnosis of a fetus with a rare karyotype of 47,XX, + 15/46,XX. Second trimester amniocentesis was performed for advanced maternal age. Fetal ultrasound revealed a hypoplastic right ventricle and intrauterine growth retardation (IUGR). The rest of the fetal anatomy was within normal limits. A mosaic karyotype of 47,XX, + 15/46,XX was observed. The couple interrupted the pregnancy at 19 weeks by dilation and suction evacuation. Careful evaluation of multiple pieces of fetal parts and placenta revealed one abnormal finding: a single umbilical artery. Cytogenetic metaphase and fluorescent in situ hybridization (FISH) interphase analyses of cells from fetal lung, heart, placenta, and skin revealed the presence of the trisomic line in all tissues. Molecular analysis demonstrated that the origin of the extra chromosome 15 was maternal, the error most likely occurred in meiosis I and the diploid line was of biparental inheritance. This case report discusses the associated findings in this fetus and reviews the literature describing other cases of mosaic trisomy 15.

Amniocentesis↗

PubMed1998

Sinorhizobium fredii and Sinorhizobium meliloti produce structurally conserved lipopolysaccharides and strain-specific K antigens.

Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens) may influence the interaction of rhizobia with their specific hosts; therefore, we conducted a comparative analysis of Sinorhizobium fredii and Sinorhizobium meliloti, which are genetically related, yet symbiotically distinct, nitrogen-fixing microsymbionts of legumes. We found that both species typically produce strain-specific K antigens that consist of 3-deoxy-D-manno-2-octulosonic acid (Kdo), or other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and hexoses. The K antigens of each strain are distinguished by glycosyl composition, anomeric configuration, acetylation, and molecular weight distribution. One consistent difference between the K antigens of S. fredii and those of S. meliloti is the presence of N-acetyl groups in the polysaccharides of the latter. In contrast to the K antigens, the LPS of Sinorhizobium spp. are major common antigens. Rough (R) LPS is the predominant form of LPS produced by cultured cells, and some strains release almost no detectable smooth (S) LPS upon extraction. Sinorhizobium spp. are delineated into two major RLPS core serogroups, which do not correspond to species (i.e., host range). The O antigens of the SLPS, when present, have similar degrees of polymerization and appear to be structurally conserved throughout the genus. Interestingly, one strain was found to be distinct from all others: S. fredii HH303 produces a unique K antigen, which contains galacturonic acid and rhamnose, and the RLPS did not fall into either of the RLPS core serogroups. The results of this study indicate that the conserved S- and RLPS of Sinorhizobium spp. lack the structural information necessary to influence host specificity, whereas the variable K antigens may affect strain-cultivar interactions.

Antigens, Bacterial↗

PubMed1997

Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A.

Natriuretic peptides, produced in the heart, bind to the natriuretic peptide receptor A (NPRA) and cause vasodilation and natriuresis important in the regulation of blood pressure. We here report that mice lacking a functional Npr1 gene coding for NPRA have elevated blood pressures and hearts exhibiting marked hypertrophy with interstitial fibrosis resembling that seen in human hypertensive heart disease. Echocardiographic evaluation of the mice demonstrated a compensated state of systemic hypertension in which cardiac hypertrophy and dilatation are evident but with no reduction in ventricular performance. Nevertheless, sudden death, with morphologic evidence indicative in some animals of congestive heart failure and in others of aortic dissection, occurred in all 15 male mice lacking Npr1 before 6 months of age, and in one of 16 females in our study. Thus complete absence of NPRA causes hypertension in mice and leads to cardiac hypertrophy and, particularly in males, lethal vascular events similar to those seen in untreated human hypertensive patients.

Animals↗

PubMed1997

Identification of the region in actin-binding protein that binds to the cytoplasmic domain of glycoprotein IBalpha.

Actin-binding protein (ABP-280) is a component of the submembranous cytoskeleton and interacts with the glycoprotein (GP) Ibalpha subunit of the GP Ib-IX complex in platelets. In the present studies, we have identified the binding site for GP Ibalpha in ABP-280. A melanoma cell line lacking ABP-280 was stably transfected with the cDNAs coding for GP Ib-IX, then transiently transfected with cDNA coding for various carboxyl-truncates of ABP-280. Immunocapture assays and co-immunoprecipitation experiments from detergent-lysed cells showed that deletion of the carboxyl-terminal repeats 20-24 of ABP-280 had no effect on GP Ib-IX binding, but deletion of residues 2099 through 2136 within repeat 19 abolished binding. In the yeast two-hybrid system, an ABP-280 fragment comprising repeats 17-19 bound GP Ibalpha. Deletion from either end abolished binding. Individual or multiple repeats of ABP-280 were expressed as fusion protein in bacteria and purified; structural folding was evaluated, and binding to GP Ib-IX was assessed. Binding depended on the presence of repeats 17-19. None of the individual repeats were able to bind to GP Ib-IX. These findings demonstrate that residues 1850-2136 comprising repeats 17-19 contain the binding site for GP Ib-IX.

Binding Sites↗

PubMed1996

Four sibs with arterial tortuosity: description and review of the literature.

We described four offspring of a consanguineous couple with arterial tortuosity "syndrome" (ATS). The affected children had extensive arterial involvement although the clinical presentations were quite variable. Clinical manifestations included cutis laxa or soft/thin skin, joint laxity or contractures, and arachnodactyly. Aortic tortuosity and pulmonary artery aneurysms with or without peripheral stenoses were demonstrated in all four sibs. All three males had inguinal hernias. Inconsistent facial anomalies were downslanting palpebral tissues, beaked nose, micrognathia, and high-arched palate. Results of collagen type I and type III biosynthesis studies were normal on skin fibroblasts. Histologic findings on autopsy of one affected child showed arterial changes with disruption of elastic fibers of the media and fragmentation of the internal elastic membrane as well as mucosal and transmural necrosis of the stomach, small bowel, colon, and extensive necrosis of the liver. Coronary artery involvement was also seen in this child as well as biventricular hypertrophy. We conclude that ATS is an autosomal recessive connective tissue condition associated with diffuse arterial changes and involvement of the skin, joints, and other organs.

Angiography↗

PubMed1996

The cytoplasmic domain of the alpha-subunit of glycoprotein (GP) Ib mediates attachment of the entire GP Ib-IX complex to the cytoskeleton and regulates von Willebrand factor-induced changes in cell morphology.

The glycoprotein (GP) Ib-IX complex is one of the major platelet membrane glycoproteins. Its extracellular domain binds von Willebrand factor at a site of injury, an interaction that leads to activation of intracellular pathways. Its intracellular domain associates tightly with the platelet cytoskeleton through actin-binding protein. The goal of the present study was to investigate the role of the cytoplasmic domain of the GP Ib-IX complex and its interaction with the cytoskeleton. Cultured cells were transfected with the cDNAs coding for GP Ib(beta), GP IX, and full-length or truncated forms of GP Ib(alpha). Western blots of detergent-insoluble fractions of Triton X-100-lysed cells showed that deletion of amino acids Trp-570 to Ser-590 from the cytoplasmic domain of GP IB(alpha) abolished the interaction of the entire GP Ib-IX complex with the cytoskeleton. Truncated GP Ib(alpha) that was unable to associate with the cytoskeleton retained its ability to associate with GP Ib(beta), to be inserted into the membrane, and to bind von Willebrand factor. Cells expressing GP Ib(alpha) changed their shape following adhesion to immobilized von Willebrand factor. Cells expressing truncated GP Ib(alpha) also changed their shape following adhesion but showed a very different morphology as compared to cells expressing full-length GP Ib(alpha). These results show that GP Ib-IX-von Willebrand factor interactions lead to cytoskeletal reorganizations, that the cytoplasmic domain of GP Ib(alpha) regulates these reorganizations, and that the cytoplasmic domain of GP Ib(alpha) is absolutely required for attachment of the GP Ib-IX complex to the cytoskeleton.

Amino Acid Sequence↗

PubMed1996

Identification of a binding sequence for the 14-3-3 protein within the cytoplasmic domain of the adhesion receptor, platelet glycoprotein Ib alpha.

The zeta-form 14-3-3 protein (14-3-3zeta) regulates protein kinases and interacts with several signaling molecules. We reported previously that a platelet adhesion receptor, glycoprotein (GP) Ib-IX, was associated with a 29-kDa protein with partial sequences identical to 14-3-3zeta. In this study, the interaction between GPIb-IX and recombinant 14-3-3zeta is reconstituted. Further, we show that the 14-3-3zeta binding site in GPIb is within a 15 residue sequence at the C terminus of GPIb-alpha, as indicated by antibody inhibition and direct binding of 14-3-3zeta to synthetic GPIb-alpha cytoplasmic domain peptides. The 14-3-3zeta binds to recombinant wild type GPIb-IX but not to the GPIb-alpha mutants lacking C-terminal 5 or more residues, suggesting that the C-terminal 5 residues of GPIb-alpha are critical. Similarity between the GPIb-alpha C-terminal sequence and the serine-rich regions of Raf and Bcr kinases suggests a possible serine-rich recognition motif for the 14-3-3 protein.

14-3-3 Proteins↗

PubMed1996

The platelet cytoskeleton stabilizes the interaction between alphaIIbbeta3 and its ligand and induces selective movements of ligand-occupied integrin.

Previously, we showed that a subpopulation of the major platelet integrin, alphaIIbbeta3, co-sediments from detergent lysates with talin and other membrane skeleton proteins. Once alphaIIbbeta3 has bound adhesive ligand in a platelet aggregate, the detergent-insoluble alphaIIbbeta3 redistributes (along with the detergent-insoluble membrane skeleton proteins and a variety of signaling molecules) to a fraction that contains cytoplasmic actin filaments. Concomitantly, certain signaling molecules are activated. The present study shows that, in intact platelets, alphaIIbbeta3 forms clusters when occupied by ligand and is selectively moved into the open canalicular system; alphaIIbbeta3 that has not bound ligand remains diffusely distributed at the periphery of the cell. When cytoplasmic actin filaments are depolymerized by cytochalasins, the ability of alphaIIbbeta3 to bind ligand is decreased, and the movement of ligand-occupied alphaIIbbeta3 is prevented. Together with the previous findings, these results suggest that (i) membrane skeleton-associated alphaIIbbeta3 is selectively induced to bind ligand in activated platelets, (ii) ligand-induced transmembrane signaling causes an altered association of membrane skeleton-associated alphaIIbbeta3 with the cytoplasmic component of the cytoskeleton, (iii) ligand-induced cytoskeletal reorganizations stabilize the interaction between ligand and integrin, and (iv) ligand-occupancy triggers cytoskeletal reorganizations that result in selective movements of occupied ligand.

Actins↗

PubMed1996

Conduction velocity in the forearm segment of the median nerve in patients with impaired conduction through the carpal tunnel.

Conduction velocity, in the forearm segment of the median nerve, has been investigated in a group of patients with carpal tunnel syndrome. Motor conduction velocity was reduced below 50 m/s in 18% of the sample. Although a correlation between severity of compression and forearm motor conduction velocity was demonstrated, it was weak (r = 0.51) and slow forearm velocities occurred in some patients who had only a minor abnormality at the carpal tunnel itself. In patients with uncomplicated carpal tunnel syndrome, slow motor conduction in the forearm segment of the median nerve was accompanied by only a small reduction (approx. 5 m/s) in mixed nerve conduction velocity. By contrast, in patients whose abnormality at the carpal tunnel was accompanied by evidence of a more widespread sensorimotor neuropathy, mixed nerve conduction velocity was reduced in parallel with the change in motor conduction velocity.

Adult↗

PubMed1996

Platelet activation: new aspects.

Work on growth factor receptors in other cells has led to the concept that tyrosine phosphorylation of receptor cytoplasmic domains leads to recruitment of signaling molecules to appropriate sub-membrane locations. Complexes of molecules are assembled through structural motifs that mediate phosphotyrosine-SH2 domain and SH3-proline-rich domain interactions. These interactions lead to activation of signaling molecules, especially activation of members of the Ras superfamily that in turn mediate downstream effects on gene regulation, vesicle movement, and cytoskeletal reorganizations. In platelets, signaling occurs primarily through members of the seven transmembrane-hererotrimeric G-protein coupled family of receptors and through adhesion receptors. The most rapid are of growth in recent years has been the realization that signaling through both G-protein coupled receptors and adhesion receptors is dependent on tyrosine kinase activation, recruitment of complexes of SH2, SH3, and proline-rich signaling molecules to appropriate locations in the cell, and on activation of members of the Ras superfamily of proteins. Because neither G-protein coupled receptors nor adhesion receptors have intrinsic tyrosine kinase activity, mechanisms presumably exist for using non-receptor kinases to initiate tyrosine phosphorylation reactions that in turn lead to the recruitment of signaling molecules. As discussed in this article, many tyrosine kinases exist in platelets and some of these are known to be tyrosine phosphorylated and activated following platelet activation. The theme that is emerging is that these tyrosine kinases may serve to phosphorylate submembranous proteins including receptors for cytoplasmic domains or components of the submembranous cytoskeleton of adhesion-receptor cytoskeleton complexes that can then recruit and activate appropriate signaling molecules. The challenge in future years will be to identity the way in which the activation of tyrosine kinase(s) is induced by receptor activation, identify the tyrosine kinase(s) involved, and identify the way in which specific members of the Ras superfamily activate downstream effectors to induce the responses of platelets to activation.

Animals↗

PubMed1995

Infant with mos45,x/46,XY/47,XYY/48,XYYY: genetic and clinical findings.

We describe an infant with mos45,X/46,XY/47,XYY/48,XYYY who presented with ambiguous genitalia. Her phenotype was also remarkable for minor ear and eye anomalies and coarctation of the aorta with bicuspid aortic valve. Laparoscopy revealed bilateral Fallopian tubes and a left infantile testis with epididymis. Chromosomal analyses of blood, skin, aorta, right Fallopian tube, and left gonadal tissue showed mos45,X/46,XY/47,XYY/48,XYYY. The 46,XY cell line was identified with routine trypsin-Giemsa banding only in cultured cells from an aortic biopsy. Fluorescence in-situ hybridization (FISH) was utilized to identify the presence of 46,XY cells in other tissues. The clinical manifestations of this patient are discussed and compared with those of similar cases of Y chromosome aneuploidy. To our knowledge, this is the first report of a patient with this unusual karyotype.

Aneuploidy↗

PubMed1995

Dystrophin-related protein in the platelet membrane skeleton. Integrin-induced change in detergent-insolubility and cleavage by calpain in aggregating platelets.

The platelet membrane is lined with a membrane skeleton that associates with transmembrane adhesion receptors and is thought to play a role in regulating the stability of the membrane, distribution and function of adhesive receptors, and adhesive receptor-induced transmembrane signaling. When platelets are lysed with Triton X-100, cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 x g) but the membrane skeleton requires 100,000 x g. The present study shows that DRP (dystrophin-related protein) sediments from lysed platelets along with membrane skeleton proteins. Sedimentation results from association with the membrane skeleton because DRP was released into the detergent-soluble fraction when actin filaments were depolymerized. Interaction of fibrinogen with the integrin alpha IIb beta 3 induces platelet aggregation, transmembrane signaling, and the formation of integrin-rich cytoskeletal complexes that can be sedimented from detergent lysates at low g-forces. Like other membrane skeleton proteins, DRP redistributed from the high-speed pellet to the integrin-rich low-speed pellet of aggregating platelets. One of the signaling enzymes that is activated following alpha IIb beta 3-ligand interactions in a platelet aggregate is calpain; DRP was cleaved by calpain to generate an approximately 140-kDa fragment that remained associated with the low-speed detergent-insoluble fraction. These studies show that DRP is part of the platelet membrane skeleton and indicate that DRP participates in the cytoskeletal reorganizations resulting from signal transmission between extracellular adhesive ligand and the interior of the cell.

Amino Acid Sequence↗

PubMed1995

Interaction of platelet glycoprotein V with glycoprotein Ib-IX regulates expression of the glycoproteins and binding of von Willebrand factor to glycoprotein Ib-IX in transfected cells.

The goal of the present study was to determine whether platelet glycoprotein (GP) V interacts directly with the von Willebrand factor receptor GP Ib-IX and, if so, whether it affects the expression and function of this receptor. A melanoma cell line that does not contain actin-binding protein was transfected with the cDNAs coding for GP V and for each of the three subunits of GP Ib-IX. GP V co-immunoprecipitated and co-localized with GP Ib-IX. Although GP V could be expressed in the absence of GP Ib-IX, the amount incorporated in the membrane was markedly increased when GP Ib-IX was present. Similarly, there was an enhanced expression of GP Ib-IX on the cell surface in the presence of GP V. The binding affinity of botrocetin-induced von Willebrand factor to GP Ib-IX was unaffected by the presence or absence of GP V. However, the binding capacity was increased by the presence of GP V. We conclude that GP V interacts directly with GP Ib-IX, that GP V must associate with GP Ib-IX to be efficiently expressed in the membrane, and that GP V increases the binding capacity of the cells for von Willebrand factor by enhancing the surface expression of the GP Ib-IX complex.

Antibodies, Monoclonal↗

PubMed1995

Multiple peptide synthesis.

The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used, only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where extreme high purity or very long peptides are required.

Automation↗

PubMed1994

Transmembrane signaling across the platelet integrin glycoprotein IIb-IIIa.

The platelet membrane is lined by a membrane skeleton, which in turn appears to be associated with underlying cytoplasmic actin filaments. Glycoprotein IIb-IIIa appears to associate with the membrane skeleton in unstimulated platelets. Upon platelet activation, unidentified intracellular signals cause GP IIb-IIIa to become competent to bind adhesive ligand. We suggest that the membrane skeleton may play a role in allowing this inside-out signaling. Signaling molecules that appear to associate with the membrane skeleton in unstimulated platelets include pp60c-src, pp62c-yes, and GAP. Preliminary evidence suggests that components of the membrane skeleton may become phosphorylated on tyrosine residues prior to GP IIb-IIIa-ligand interactions. Once GP IIb-IIIa binds adhesive ligand in a platelet aggregate, there is signaling in the opposite direction. One consequence of the outside-in transmembrane signaling is that the membrane skeleton becomes more tightly associated with the underlying actin filaments as focal contact-like structures form. Proteins that accumulate in these focal contact-like structures with a time course identical to that of GP IIb-IIIa and in a GP IIb-IIIa-dependent manner include talin, vinculin, and spectrin. Signaling molecules that accumulate in the focal contact-like structures include pp60c-src, pp62c-yes, phosphoinositide 3-kinase, and protein kinase C. These are potential candidates for the enzymes that mediate the ligand-induced transmembrane signaling. Another enzyme involved in the ligand-induced signaling is calpain. This enzyme is activated as a consequence of ligand-GP IIb-IIIa interactions and cleaves components of the membrane skeleton. Future experiments will be needed to identify other signaling enzymes activated as a consequence of GP IIb-IIIa interactions and to determine which ones are responsible for inducing the cytoskeletal reorganizations that occur in platelets and other cells when integrins bind their adhesive ligands.

Animals↗