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V Bennett

Publications and source records attributed to V Bennett.

At least 55 records · Page 3Linked to original sources
PubMed1996

Molecular composition of the node of Ranvier: identification of ankyrin-binding cell adhesion molecules neurofascin (mucin+/third FNIII domain-) and NrCAM at nodal axon segments.

Neurofascin, NrCAM, L1, and NgCAM are a family of Ig/FNIII cell adhesion molecules that share ankyrin-binding activity in their cytoplasmic domains, and are candidates to form membrane-spanning complexes with members of the ankyrin family of spectrin-binding proteins in a variety of cellular contexts in the nervous system. Specialized forms of ankyrin, 270 kD and/or 480 kD ankyrinG are components of the membrane undercoat of axons at the node of Ranvier. This paper focuses on definition of the isoforms of ankyrin-binding cell adhesion molecules localized with ankyrinG at the nodal axon segment. The exon usage of two major forms of neurofascin was determined by isolation of full-length cDNAs and used to prepare isoform-specific antibodies. An isoform of neurofascin containing a mucin-like domain and lacking the third FNIII domain was concentrated at axon initial segments and colocalized at nodes of Ranvier with ankyrinG and the voltage-dependent sodium channel. An alternative form of neurofascin lacking the mucin-like domain and containing the third FNIII domain was present in unmyelinated axons. The antibody initially raised against neurofascin was used to screen a rat brain cDNA expression library. In addition to neurofascin, this screen yielded a clone with 80% sequence identity to NrCAM from chicken. The sequences of two full-length cDNAs are presented. NrCAM is most closely related to neurofascin among the other members of the L1/neurofascin/NgCAM family, with over 70% identity between cytoplasmic domains. NrCAM, visualized with antibodies specific for the ecto-domain, also was found to be coexpressed with neurofascin at nodes of Ranvier and at axon initial segments. This is the first characterization of defined neuronal cell adhesion molecules localized to axonal membranes at the node of Ranvier of myelinated axons.

Alternative Splicing↗
PubMed1995

Mechanism for binding site diversity on ankyrin. Comparison of binding sites on ankyrin for neurofascin and the Cl-/HCO3- anion exchanger.

Ankyrins are a family of spectrin-binding proteins that associate with at least seven distinct membrane proteins, including ion transporters and cell adhesion molecules. The membrane-binding domain of ankyrin is comprised of a tandem array of 24 ANK repeats organized into four 6-repeat folding domains. Tandem arrays of ANK repeats have been proposed to mediate protein interactions in a variety of proteins including factors involved in the regulation of transcription and the cell cycle. This report provides several new insights into the versatility of ANK repeats of ankyrin in protein recognition, using neurofascin and the Cl-/HCO3- anion exchanger as model ligands and ankyrinR as the prototypic ankyrin. Different combinations of ANK repeat domains from this ankyrin form two distinct, high affinity binding sites for neurofascin. One site requires both repeat domains 3 and 4. The other site involves both repeat domains 2 and 3, although domain 2 has significant activity alone. The sites appear to be independent with Kd values of 3 and 14 nM, respectively. Both the Cl-/HCO3- anion exchanger and neurofascin can interact simultaneously with repeat domains 3 and 4, because neurofascin is unable to displace binding of the anion exchanger cytoplasmic domain to domains 3 and 4, despite having a 3-5-fold higher affinity. These results demonstrate two levels of diversity in the binding sites on ankyrin: one resulting from different combinations of ANK repeat domains and another from different determinants within the same combination of repeat domains. One consequence of this diversity is that ankyrin can accommodate two neurofascin molecules as well as the anion exchanger through interactions mediated by ANK repeats. The ability of ankyrin to simultaneously associate with multiple types of membrane proteins is an unanticipated finding with implications for the assembly of integral membrane proteins into specialized regions of the plasma membrane.

Ankyrins↗
PubMed1995

The ANK repeats of erythrocyte ankyrin form two distinct but cooperative binding sites for the erythrocyte anion exchanger.

The 24 ANK repeats of the membrane-binding domain of ankyrin form four folded subdomains of six ANK repeats each. These four repeat subdomains mediate interactions with at least seven different families of membrane proteins. In the erythrocyte, the main membrane target of ankyrin is the Cl-/HCO3- anion exchanger. This report presents the first evidence that ankyrin contains two separate binding sites for anion exchanger dimers. One site utilizes repeat subdomain two (repeats 7-12) while the other requires both repeat subdomains three and four (repeats 13-24). The two sites are positively coupled with a Hill coefficient of 1.4. Since the anion exchanger exists as a dimer in the membrane, the presence of two binding sites on ankyrin allows ankyrin to interact with four anion exchangers simultaneously. These findings provide a direct demonstration of the versatility of ANK repeats in protein recognition, and have important implications for the organization of ankyrin-linked integral membrane proteins in erythrocytes as well as other cells.

Amino Acid Sequence↗
PubMed1995

Adducin: a physical model with implications for function in assembly of spectrin-actin complexes.

Adducin binds to spectrin-actin complexes, promotes association of spectrin with actin, and is subject to regulation by calmodulin as well as protein kinases A and C. Adducin is a heteromer comprised of homologous alpha and beta-subunits with an NH2-terminal protease-resistant head domain, connected by a neck region to a COOH-terminal hydrophilic, protease-sensitive region. This study provides evidence that adducin in solution is a mixture of heterodimers and tetramers. CD spectroscopy of COOH-terminal domains of alpha- and beta-adducin bacterial recombinants provides direct evidence for an unstructured random coil configuration. Cross-linking, proteolysis, and blot-binding experiments suggest a model for the adducin tetramer in which four head domains contact one another to form a globular core with extended interacting alpha- and beta-adducin tails. The site for binding to spectrin-actin complexes on adducin was identified as the COOH-terminal tail of both the alpha- and beta-adducin subunits. The capacity of native adducin to recruit spectrin to actin filaments is similar to that of adducin tail domains. Thus, adducin tail domains alone are sufficient to interact with F-actin and a single spectrin and to recruit additional spectrin molecules to the ternary complex.

Actins↗
PubMed1995

Chromosomal localization of the ankyrinG gene (ANK3/Ank3) to human 10q21 and mouse 10.

The ankyrin3 gene encodes a novel form of ankyrin, AnkyrinG, expressed in multiple tissues but characteristically present at the axonal initial segment and nodes of Ranvier of neurons in the central and peripheral nervous systems. We have localized ANK3 to human Chromosome 10q21 by fluorescence in situ hybridization. The position of the murine homologue was determined by intersubspecific backcross analysis, mapping Ank3 to mouse Chromosome 10, between microsatellite marker D10Mit31 and the Bcr gene. This interval is known to comprise a region syntenic to human 10q. The localization of ANK3 is a preliminary step in identifying neurological disorders potentially associated with the gene.

Animals↗
PubMed1995

AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier.

We have characterized a new ankyrin gene, expressed in brain and other tissues, that is subject to extensive tissue-specific alternative mRNA processing. The full-length polypeptide has a molecular mass of 480 kDa and includes a predicted globular head domain, with membrane- and spectrin-binding activities, as well as an extended "tail" domain. We term this gene ankyrinG based on its giant size and general expression. Two brain-specific isoforms of 480 kDa and 270 kDa were identified that contain a unique stretch of sequence highly enriched in serine and threonine residues immediately following the globular head domain. Antibodies against the serine-rich domain and spectrin-binding domain revealed labeling of nodes of Ranvier and axonal initial segments. Ankyrin-binding proteins also known to be localized in these specialized membrane domains include the voltage-dependent sodium channel, the sodium/potassium ATPase, sodium/calcium exchanger, and members of the neurofascin/L1 family of cell adhesion molecules. The neural-specific ankyrinG polypeptides are candidates to participate in maintenance/targeting of ion channels and cell adhesion molecules to nodes of Ranvier and axonal initial segments.

Alternative Splicing↗
PubMed1995

Late intracytoplasmic sperm injection in unexpected failed fertilization in vitro: diagnostic or therapeutic?

OBJECTIVE: To evaluate fertilization potential of 24-hour-old unfertilized oocytes using intracytoplasmic sperm injection and the pregnancy potential of resultant embryos. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London, United Kingdom. PATIENTS: Fifteen patients with a history of infertility who underwent treatment with IVF and showed failure of fertilization on the day after oocyte retrieval. INTERVENTION: Assisted fertilization with intracytoplasmic sperm injection was carried out at 24 hours after oocyte retrieval. RESULTS: A total of 121 metaphase II oocytes were subjected to intracytoplasmic sperm injection. Of these, 9 were damaged (7%), 2 were polyploidic (2%), and 58 showed normal fertilization (48%). Of the latter, 47 cleaved normally (81%). Forty embryos were transferred and three were cryopreserved. One patient conceived (7%) but in this case only one of three embryos transferred was from intracytoplasmic sperm injection. CONCLUSION: Late (24 hours) intracytoplasmic sperm injection can give good fertilization and cleavage rates but the potential of the generated embryos to achieve pregnancy seems to be low.

Adult↗
PubMed1995

Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia.

OBJECTIVE: To evaluate the recovery rate of spermatozoa from the epididymis using a percutaneous aspiration technique and to examine the fertilization rate after intracytoplasmic sperm injection. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London. SUBJECTS: Twenty patients with obstructive azoospermia who each had an attempt at IVF. The sperm used for intracytoplasmic sperm injection was retrieved by percutaneous epididymal sperm aspiration in 16 patients. In one patient, microepididymal sperm aspiration was performed in addition because the quality of the sperm obtained by percutaneous epididymal sperm aspiration was not considered suitable for microinjection. In the remaining three patients, neither percutaneous epididymal sperm aspiration nor microepididymal sperm aspiration resulted in the recovery of sperm, which was obtained by testicular biopsy in one of them. INTERVENTION: Assisted fertilization with intracytoplasmic sperm injection. MAIN OUTCOME MEASURES: Normal fertilization and pregnancy rates. RESULTS: A total of 179 eggs were collected and 157 subsequently were microinjected. Normal fertilization occurred in 22 oocytes (14%) and the total number of embryos cleaved was 30. Twelve patients underwent ET in which three conceived (pregnancy rate 25% per transfer). The implantation rate was 10% and failed fertilization occurred in four cycles. CONCLUSION: Percutaneous epididymal sperm aspiration can be used successfully to recover sperm in men with obstructive azoospermia for use in assisted fertilization IVF cycles. The technique is simple, effective, and less traumatic compared with an open microsurgical operation.

Adult↗
PubMed1995

Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells.

Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G-spectrin, although alpha-spectrin was still present. Beta G-spectrin-deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin-associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E-cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)-ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.

Ankyrins↗
PubMed1994

Ankyrin binding activity shared by the neurofascin/L1/NrCAM family of nervous system cell adhesion molecules.

Neurofascin, L1, NrCAM, NgCAM, and neuroglian are membrane-spanning cell adhesion molecules with conserved cytoplasmic domains that are believed to play important roles in development of the nervous system. This report presents biochemical evidence that the cytoplasmic domains of these molecules associate directly with ankyrins, a family of spectrin-binding proteins located on the cytoplasmic surface of specialized plasma membrane domains. Rat neurofascin and NrCAM together comprise over 0.5% of the membrane protein in adult brain tissue. Linkage of these ankyrin-binding cell adhesion molecules to spectrin-based structures may provide a major class of membrane-cytoskeletal connections in adult brain as well as earlier stages of development.

Amino Acid Sequence↗
PubMed1994

Identification of two regions of beta G spectrin that bind to distinct sites in brain membranes.

This study analyzed the complex interactions of intact spectrin with bovine brain membranes by evaluating membrane associations of defined regions of beta G spectrin, the subunit responsible for high affinity membrane binding. Two regions of beta G spectrin were expressed in bacteria and demonstrated to contain fully functional binding site(s) for a subset of spectrin-binding sites in brain membranes depleted of peripheral proteins. One region, located near the NH2 terminus, was comprised of 106-residue repeats and required repeats 2-7 for full activity. The other binding domain was located at the COOH terminus, which is the most variable between beta G and beta R spectrins, is distinct from the 106-residue repeats, and contains a pleckstrin homology domain. NH2-terminal beta spectrin polypeptides interacted with a membrane site(s) that recognized both brain and erythrocyte isoforms of spectrin, was inhibited by calcium/calmodulin, and was not blocked by the COOH-terminal polypeptide. The COOH-terminal region associated with a membrane site(s) that was specific for brain spectrin, was not inhibited by calcium/calmodulin, and was not blocked by the NH2-terminal polypeptide. These observations demonstrate membrane association of spectrin with at least two independent sites, which differ with regard to regulation by calcium/calmodulin and in selectivity for spectrin isoforms.

Animals↗
PubMed1994

Experience with subzonal insemination (SUZI) and intracytoplasmic sperm injection (ICSI) on unfertilized aged human oocytes.

OBJECTIVE: The aim of this study was to assess the fertilizability of unfertilized aged human oocytes from failed in vitro fertilization (IVF) cycles using SUZI and ICSI. METHODS: A total of 363 oocytes which showed no fertilization after conventional IVF was subjected to assisted fertilization using SUZI or ICSI. The microinjected oocytes which were derived from 72 patients undergoing their first IVF treatment had an intact polar body and no signs of degeneration. SUZI was carried out in 265 oocytes and ICSI in the remaining 98. RESULTS: Significantly more oocytes were damaged after ICSI (9 vs 0.3%, P < 0.01). Normal fertilization rates were higher at 24 hr in both groups and occurred more frequently after ICSI, although the difference did not reach statistical significance. Abnormal fertilization occurred significantly more often after SUZI at 48 hr (P < 0.005), but not at 24 hr. Cleavage rates were significantly higher after ICSI (94.4 vs 57.1%, P < 0.025) at 24 hr, but this was not observed at 48 hr, although the ICSI group still showed better cleavage rates (33.3 vs 19.1%). There was no difference in embryo quality in either group. CONCLUSIONS: Our results indicate that micromanipulation rather than reinsemination should be carried out on unfertilized human oocytes from failed IVF attempts. Both techniques can be used to achieve fertilization which occurs more often after ICSI. However, the trauma from the former technique on the microinjected oocytes may impair the potential of the generated embryos to achieve pregnancy compared to SUZI. Prospective randomized trials are necessary to address the problem.

Adult↗
PubMed1993

The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats.

Ankyrin repeats are a 33-amino acid motif present in a number of proteins of diverse functions including transcription factors, cell differentiation molecules, and structural proteins. This motif has been shown to mediate protein interactions in the case of ankyrin as well as several other repeat-bearing proteins. In ankyrin, 24 tandemly arrayed repeats are arranged to form a globular, membrane-binding domain. This report provides evidence that the repeats in this domain fold into four independently folded subdomains of six repeats each. Limited proteolytic digestions of defined regions of the membrane-binding domain identified protease-sensitive sites, which divided this domain into subdomains of approximately six repeats each. Hydrodynamic measurements and circular dichroism spectroscopy of expressed subdomains confirmed that these six-repeat regions exist as folded, globular structures. The requirement of a complete set of six repeats for proper folding was determined using a series of protein constructs, which sequentially deleted repeats from the last subdomain. Deletion of even one repeat resulted in a 40% loss of alpha-helicity. Deletions removing three or more repeats abolished the helical signal completely. The spherical shapes of the intact domain and of the subdomains (inferred from hydrodynamic values) suggest that the four subdomains are organized in either a tetrahedral or square planar configuration. Two six-repeat subdomains were found to be required for high affinity association with the anion exchanger, suggesting that at least some of the protein interactions mediated by ankyrin repeats involve multiple subdomains.

Amino Acid Sequence↗
PubMed1993

The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin.

Na+-Ca2+ exchange is the major pathway of Ca2+ efflux during excitation-contraction coupling in cardiac muscle. The Na+-Ca2+ exchanger is present in cardiac transverse tubules with an apparent high density (Frank, J.S., Mottino, G., Reid, D., Molday, R. S., and Philipson, K.D. (1992) J. Cell Biol. 117, 337-345). The mechanism for this localization is unknown but may involve interactions with the cytoskeleton. In the present study, we examined the interaction of the Na+-Ca2+ exchanger with the cytoskeletal protein ankyrin. On immunoblots of isolated canine cardiac sarcolemma, an antibody raised against purified rabbit red blood cell-ankyrin (RBC-ankyrin) recognized a 220-kDa protein, which is the same size as RBC-ankyrin. Alkaline extraction of sarcolemma removed this protein. The Na+-Ca2+ exchange protein, purified from recombinant baculovirus-infected insect cells, bound 125I-labeled-RBC-ankyrin with a KD of 42 +/- 3 nm. 125I-RBC-ankyrin was co-precipitated by antibodies to the Na+-Ca2+ exchanger after preincubation with solubilized cardiac sarcolemma. Myocardial ankyrin could be localized to both surface and T-tubular sarcolemma by immunofluorescence techniques. These results demonstrate that the cardiac Na+-Ca2+ exchanger binds ankyrin with high affinity. This interaction may be important for localizing the Na+-Ca2+ exchanger to specific domains of the sarcolemma.

Animals↗
PubMed1993

From anemia to cerebellar dysfunction. A review of the ankyrin gene family.

The focus of this review is on the ankyrin gene family, key elements in the interaction of the spectrin-based membrane skeleton with the plasma membrane in a variety of tissues and multicellular organisms. The structure/function relationships of ankyrin molecules are reviewed, illustrating how these proteins are uniquely suited to serve as adaptors between the membrane skeleton and a number of integral membrane proteins. Advances in the understanding of ankyrin biology in the brain are discussed and used to show how ankyrins may be involved in the establishment and/or maintenance of specialized plasma membrane domains. Finally, recent research in hematological and neurological disorders are reviewed, suggesting that ankyrins have a role in the development of human disease.

Animals↗