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S Conner

Publications and source records attributed to S Conner.

15 recordsLinked to original sources
PubMed2001

The biology of cortical granules.

An egg-that took weeks to months to make in the adult-can be extraordinarily transformed within minutes during its fertilization. This review will focus on the molecular biology of the specialized secretory vesicles of fertilization, the cortical granules. We will discuss their role in the fertilization process, their contents, how they are made, and the molecular mechanisms that regulate their secretion at fertilization. This population of secretory vesicles has inherent interest for our understanding of the fertilization process. In addition, they have import because they enhance our understanding of the basic processes of secretory vesicle construction and regulation, since oocytes across species utilize this vesicle type. Here, we examine diverse animals in a comparative approach to help us understand how these vesicles function throughout phylogeny and to establish conserved themes of function.

Amino Acid Sequence↗
PubMed2000

SFE1, a constituent of the fertilization envelope in the sea urchin is made by oocytes and contains low-density lipoprotein-receptor-like repeats.

At fertilization in most animals, cortical granules of the egg or oocyte secrete their contents, whose function it is to modify the extracellular matrix. This modified matrix then participates in the block to polyspermy and protection for early embryonic development. In the sea urchin, contents of the cortical granules are secreted within 30 sec of insemination. Several of these content proteins then bind to the nascent vitelline layer of the egg and lift off the cell surface to form a stable, impervious, fertilization envelope. At least six major proteins are present in the envelope, and recently we have identified cDNA clones of two, ovoperoxidase, and SFE9. Here we report on the identification and characterization of SFE1, a constituent of the fertilization envelope of the sea urchin Strongylocentrotus purpuratus, that has revealing characteristics of how the envelope might form and what protein interaction domains might predominate. We present the largest cDNA sequence we were able to identify representing approximately two thirds of the predicted protein coding region. The C-terminal half of the cognate SFE1 protein contains two different amino acid repeat motifs: a cysteine-rich (15%) motif of 40 amino acids that is tandemly repeated 22 times and is followed by a serine/threonine-rich (38%) repeat of 63 amino acids that is tandemly repeated 3.5 times. Surprisingly, just N-terminal to the cysteine-rich repeat region is a sequence of five repeats with similarity to repeats in another cortical granule protein, SFE9, and to the motif originally identified in the receptor of low-density lipoproteins, the LDLr motif. The amino acid composition deduced from the partial SFE1 cDNA is similar also to the composition of proteoliaisin, a protein thought to tether the ovoperoxidase to the vitelline layer of the egg and thereby sequester the crosslinking activity of the ovoperoxidase to a limited population of proteins in the fertilization envelope. However, by use of monoclonal and polyclonal antibodies to SFE1 and proteoliaisin, we show here that they are distinct gene products. We also show that SFE1 is packed selectively into the cortical granules and then is crosslinked into the fertilization envelope following fertilization. In situ RNA hybridization analysis shows that the mRNA of SFE1 (9 kilobases) is present in oocytes selectively and is turned over rapidly in the oocyte following germinal vesicle breakdown. Our findings suggest that the gene encoding this major product of the egg is activated concomitantly with the other cortical granule-specific products already identified, and that a common LDLr-like motif of the fertilization envelope may reveal a structural mechanism for protein interactions in its construction.

Amino Acid Sequence↗
PubMed1998

rab3 mediates cortical granule exocytosis in the sea urchin egg.

Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking.

Amino Acid Sequence↗
PubMed1997

Members of the SNARE hypothesis are associated with cortical granule exocytosis in the sea urchin egg.

Cortical granule exocytosis is important for the block to polyspermy at fertilization in the eggs of most vertebrates and many invertebrates. Cortical granules are poised at the cell surface and exocytose in response to sperm stimulation. Following exocytosis, the cortical granule contents modify the extracellular environment of the egg, the major result of which is to block additional sperm binding. Here we show that proteins homologous to members of the SNARE hypothesis-a molecular model designed to explain the trafficking, docking, and exocytosis of vesicles in the secretory compartment-are present in eggs at the right time and place to be involved in the regulation of cortical granule exocytosis. Using polymerase chain reaction (PCR) screens we have found homologues of synaptobrevin/VAMP, syntaxin, synaptotagmin, and rab3. Antibodies generated to fusion proteins or to synthetic peptides encoded by the cloned cDNAs were used in an immunofluorescence assay to show that each of the cognate proteins are present in the cortex of the egg. A synaptobrevin/VAMP homologue appears to be specifically associated with the membrane of cortical granules before fertilization and, following cortical granule exocytosis, is incorporated into the plasma membrane of the zygote. A rab3 homologue is also associated with cortical granules specifically but, following fertilization, the protein reassociates with different, yet undefined, vesicles throughout the cytoplasm of the zygote. Homologues of synaptotagmin and syntaxin are also present at the egg cortex but, in contrast to rab3 and VAMP, appear to be associated with the plasma membrane. Following fertilization, syntaxin and tagmin remain associated with the plasma membrane and are more readily immunolabeled, presumably due to an increased accessibility of the antibodies to the target protein domains. We also show by immunoblotting experiments that the cognate proteins are of the sizes predicted for these homologues. These results suggest that at least some steps in the biology of cortical granules may be mediated by SNARE homologues, and this finding, along with the unique biology of cortical granules, should facilitate examination of specific events of the fertilization reaction and the mechanism of stimulus-dependent exocytosis.

Amino Acid Sequence↗
PubMed1995

Flow cytometric study of differentiating cultures of Bacillus subtilis.

We report on 1) the development of a flow cytometry-based technique for detecting beta-galactosidase in differentiating cultures of Bacillus subtilis and 2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5-octanolyaminofluorescein di-beta-D-galactopyranoside (C8-FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C8-FDG staining mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C8-fluorescein and PKH26 allow nonspecific C8-FDG staining in this control population to be monitored using two-color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulation genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild-type and mutant cultures early during the development process of spore formation.

Bacillus subtilis↗
PubMed1993

A quantitative study of electroporation showing a plateau in net molecular transport.

Electroporation is believed to involve a temporary structural rearrangement of lipid bilayer membranes, which results in ion and molecular transport across the membrane. The results of a quantitative study of molecular transport due to electroporation caused by a single exponential pulse are presented; transport of four molecules of different physical characteristics across erythrocyte ghost membranes is examined as a function of applied field strength. Flow cytometry is used to quantitatively measure the number of molecules transported for 10(4) to 10(5) individual ghosts for each condition. This study has four major findings: 1) Net transport first increases with field strength, but reaches a plateau at higher field strengths. Significant transport is found at or below 1 kV/cm, and transport plateaus begin at field strengths between 2 and 5 kV/cm depending on the molecule transported. 2) A single population of ghosts generally exists, but exhibits a wide distribution in the amount of molecular transport. 3) Under the conditions used, the direction of transport across the ghost membrane does not appear to affect molecular transport significantly. 4) Large numbers of ghosts may be destroyed by the electroporation procedure.

Adult↗
PubMed1993

Comparative clinical, microbiologic, and economic audit of the use of oral ciprofloxacin and parenteral antimicrobials.

OBJECTIVE: To examine the use of oral ciprofloxacin and parenteral antimicrobials in the treatment of acute infection (respiratory tract, urinary tract, blood) in hospitalized patients, with particular reference to severity of infection, outcome, and associated economic implications of each treatment. DESIGN: A prospective, multicenter comparative audit was conducted in the UK over an 18-month period. The audit was undertaken by clinical pharmacists who reviewed the antimicrobial treatment of patients with infective episodes, who were receiving or could have received oral therapy. PARTICIPANTS: Clinical pharmacists who collated the data all are employed in teaching hospitals. MAIN OUTCOME MEASURES: Patients were identified for analysis by their type of infection, severity of illness, antimicrobial prescribed, route of administration, and response to therapy. Additionally, data on costs of the antimicrobials prescribed and the supplementary costs of drug administration were calculated. RESULTS: Four hundred eighty-five patients were enrolled for analysis; 208 of the patients had respiratory tract infection, 112 had urinary tract infection, 138 had septicemia, and 27 had mixed infections. Sepsis scores were applied to 152 patients receiving oral ciprofloxacin and 333 patients receiving parenteral antimicrobials and yielded mean scores of 5.9 (SD 3.1, range 1-13) and 8.7 (SD 4.2, range 1-22), respectively. Of 485 patients, 188 were paired according to sepsis score results and route of administration. Resolution occurred in 133 patients (79 receiving oral and 54 receiving parenteral therapy), further therapy was required in 49 (11 oral and 38 parenteral), and treatment was withdrawn in 6 (4 oral and 2 parenteral). Microbiologic assessment yielded positive results in 227 patients (47 percent). Drug acquisition costs (based on 1 UK pound = 1.80 US $) per course of treatment were $47.23 (SD $38.32, range 5.40-218.70) for the oral group and $173.70 (SD 209.77, range 2.11-1021.40) for the parenteral group. Overall costs for treatment courses were $74.00 (SD $52.81, range 8.10-309.17) and $305.59 (SD $304.70, range 14.69-1345.00) for the two groups, respectively (p < 0.001). CONCLUSIONS: This study suggest that, in patients with moderate infective episodes, oral ciprofloxacin may reduce overall treatment costs compared with the use of parenteral antimicrobial agents.

Adolescent↗
PubMed1984

Gene conversion between murine class II major histocompatibility complex loci. Functional and molecular evidence from the bm 12 mutant.

The experiments presented in this study define the molecular basis of the bm 12 mutation. Initial characterization of an alloreactive T cell clone, 4.1.4, showed this clone to recognize an allodeterminant present on the E beta b and A beta bm12 chains, but not on the bm 12 parent A beta b chain. To define the extent of sequence shared between the I-E beta product and the mutant I-A beta product, we isolated a cDNA clone of the E beta b gene and determined its nucleotide sequence. Comparison of the nucleotide sequences of E beta b, A beta b, and A beta bm12 shows the the A beta bm12 gene to be identical to the E beta b gene in the region where it differs from its A beta b parent. We predict that the bm 12 mutation arose by gene conversion of this region, which spans 14 nucleotides between amino acid residues 67-71 of the mature A beta chain, from the E beta b locus to the corresponding position at the A beta b locus. Recognition of this region, which spans one of the previously defined E beta allelic "hypervariable" regions, by an alloreactive T cell clone provides the first direct evidence of the functional importance of these hypervariable regions in T cell stimulation. The identification of a gene conversion event involving one of these allelic variable regions implicates conversion as a mechanism that acts on class II beta genes to create sequence diversity in regions of Ia molecules that interact with foreign antigen or a T cell receptor, regions where protein sequence polymorphism would presumably be selected for by the expanded ability it affords the organism to mount effective immune responses against a wider variety of foreign antigens.

Animals↗
PubMed1982

Spontaneous germinal matrix and intraventricular hemorrhage in prematurely born rabbits.

Spontaneous hemorrhage into the ventricles in premature babies is a major problem, and neither its cause nor its pathogenesis is understood. A model is presented for the study of germinal matrix and intraventricular hemorrhage in the preterm rabbit. This animal is particularly suitable because, like the human, 1) the maximal growth of the brain occurs perinatally; 2) there is an abundant germinal matrix near term, and by birth this is substantially reduced; 3) there is no rete mirabile; 4) the blood flow to the brain is via the internal carotid and vertebral arteries; 5) the maturation of the lungs is completed just before term; and 6) the rabbit pup can maintain a separate existence from the dam when delivered prematurely. Eight of 64 such animals were found to have developed spontaneous germinal matrix hemorrhage with or without rupture into the ventricles. Several physiological and chemical features characteristic of the premature rabbit are presented. The hemorrhage in the lagomorph might be a paradigm of that in infants, and its study may aid in the understanding of the pathogenesis of the process.

Animals↗
PubMed1981

Changes in the water and electrolyte pattern of the brain and of the intracranial pressure during development in rabbits.

During its development the brain becomes dehydrated pari passu with growth and myelination and the electrolyte pattern changes from high sodium, low potassium to the opposite. The latter change is complete or virtually so in some species at term but in others the reversal is a postnatal occurrence. We have examined these features in rabbits of several ages and have measured the intracranial pressure at intervals during development as well. At or near both the water content of the brain is very high compared to that found later and the fetal electrolyte pattern is transitional in that the K/Na ratio is approximately one but not as high as in the mature brain. The intracranial pressure is lower in the newborn pups than it is later in development. Unlike that in mature animals the premature and young term rabbit brain is unable to affect ionic movements appropriate to the protection of the volume of the brain.

Animals↗
PubMed1975

Thermal properties of cancellous bone.

Specimens of beef bone were analyzed for thermal conductivity and thermal capacity. The thermal conductivity was about 7 X 10(-4) cal/cm/sec/degrees C, that is about the same as for other tissues. As regards thermal capacity, the determination of 0.73 cal/cm2/degrees C also was similar to that in other tissues. Both thermal conductivity and capacity were isotropic with respect to the layer of epiphyseal cartilage, and the orientation of bony trabeculae. Thermal probes were devised heated either by water or by electric current to destroy localized volumes of bone, as in surgery. To achieve necrotizing temperatures, the sizes of such probes and the times of application of heat were determined.

Animals↗