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S V Smith

Publications and source records attributed to S V Smith.

At least 37 records · Page 2Linked to original sources
PubMed1998

188Re DD-3B6/22 Fab' for use in therapy of ovarian cancer: labelling and animal studies.

A fast and high yielding method of 188Re radiolabelling DD-3B6/22 Fab' is described. An inert atmosphere [N2(g)] and ascorbic acid was essential for preparation and storage of therapeutic levels (< or =2 GBq/mg) for up to 24 h. Immunoreactivity was greater than 75%. Pharmacokinetic studies in nu/nu mice demonstrated localisation of 188Re DD-3B6/22 Fab' was equivalent and correlated well with the behaviour observed for 99mTc DD-3B6/22 Fab' used to image ovarian cancer. Excellent stability at the target site in vivo supports the potential use of 188Re DD-3B6/22 Fab' in the therapy of ovarian cancer.

Animals↗
PubMed1998

Potentiometric study of resting potential, contributing K+ channels and the onset of Na+ channel excitability in embryonic rat cortical cells.

Resting membrane potential (RMP), K+ channel contribution to RMP and the development of excitability were investigated in the entire population of acutely dissociated embryonic (E) rat cortical cells over E11-22 using a voltage-sensitive fluorescent indicator dye and flow cytometry. During the period of intense proliferation (E11-13), two cell subpopulations with distinct estimated RMPs were recorded: one polarized at approximately -70 mV and the other relatively less-polarized at approximately -40 mV. Ca2+o was critical in sustaining the RMP of the majority of less-polarized cells, while the well-polarized cells were characterized by membrane potentials exhibiting a approximately Nernstian relationship between RMP and [K+]o. Analysis of these two subpopulations revealed that > 80% of less-polarized cells were proliferative, while > 90% of well-polarized cells were postmitotic. Throughout embryonic development, the disappearance of Ca2+o-sensitive, less-polarized cells correlated with the disappearance of the proliferating population, while the appearance of the K+o-sensitive, well-polarized population correlated with the appearance of terminally postmitotic neurons, immuno-identified as BrdU-, tetanus toxin+ cells. Differentiating neurons were estimated to contain increased K+i relative to less-polarized cells, coinciding with the developmental expression of Cs+/Ba2+-sensitive and Ca2+-dependent K+ channels. Both K+ channels contributed to the RMP of well-polarized cells, which became more negative toward the end of neurogenesis. Depolarizing effects of veratridine, first observed at E11, progressively changed from Ca2+o-dependent and tetrodotoxin-insensitive to Na+o-dependent and tetrodotoxin-sensitive response by E18. The results reveal a dynamic development of RMP, contributing K+ channels and voltage-dependent Na+ channels in the developing cortex as it transforms from proliferative to primarily differentiating tissue.

Animals↗
PubMed1998

GABAergic cells and signals in CNS development.

GABA is formed primarily from decarboxylation of glutamate by a family of cytosolic and membrane-bound GAD enzymes. In the adult, GAD-derived GABA sustains the vitality of the central nervous system (CNS), since blockage of GAD rapidly leads to convulsions and death. In plants, cytosolic GAD synthesizes GABA in response to hormones and environmental stress. Since decarboxylation involves protonation, secretion of GABA serves to buffer cytosolic pH in plant cells. Families of GAD and GABAA receptor/Cl- channel transcripts and encoded proteins emerge early and seemingly everywhere during CNS development, with their abundance closely paralleling neurogenesis and peaking before birth. Micromolar GABA acts at receptor/Cl-channels to depolarize progenitor cells in the cortical neuroepithelium; it also elevates their cytosolic Ca2+ (Cac2+) levels. In some way, these effects decrease proliferation. GABA directs the migration of postmitotic neuroblasts at femtomolar concentrations and stimulates their random motility at micromolar concentrations via Ca2+ signaling mechanisms. Activation of GABAA receptors by micromolar GABA may limit motility via membrane depolarization and elevated Cac2+. These results indicate that in vitro GABA can affect embryogenesis of the CNS through effects on cell proliferation and migration. As neurons differentiate postnatally, Cl(-)-dependent depolarization disappears together with GABAergic Cac2+ signals. Physiologically occurring GABAergic signals at Cl-channels exist in tonic and transient forms. Since the former are found on progenitor cells while both are present in postmitotic neurons, mechanisms to generate transients differentiate in the latter. Surprisingly, tonic and transient forms of GABAergic signaling at Cl-channels are rapidly and smoothly interconvertible and seem to be derived from online GABA synthesis in a surface-accessible compartment of the membrane.

Animals↗
PubMed1997

Identification of a sialidase encoded in the human major histocompatibility complex.

Mammalian sialidases are important in modulating the sialic acid content of cell-surface and intracellular glycoproteins. However, the full extent of this enzyme family and the physical and biochemical properties of its individual members are unclear. We have identified a novel gene, G9, in the human major histocompatibility complex (MHC), that encodes a 415-amino acid protein sharing 21-28% sequence identity with the bacterial sialidases and containing three copies of the Asp-block motif characteristic of these enzymes. The level of sequence identity between human G9 and a cytosolic sialidase identified in rat and hamster (28-29%) is much less than would be expected for analogous proteins in these species, suggesting that G9 is distinct from the cytosolic enzyme. Expression of G9 in insect cells has confirmed that it encodes a sialidase, which shows optimal activity at pH 4.6, but appears to have limited substrate specificity. The G9 protein carries an N-terminal signal sequence and immunofluorescence staining of COS7 cells expressing recombinant G9 shows localization of this sialidase exclusively to the endoplasmic reticulum. The location of the G9 gene, within the human MHC, corresponds to that of the murine Neu-1 locus, suggesting that these are analogous genes. One of the functions attributed to Neu-1 is the up-regulation of sialidase activity during T cell activation.

Amino Acid Sequence↗
PubMed1997

Monitoring papain digestion of a monoclonal antibody by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has been used to examine the Fab, F(ab')2 and deglycosylated Fc fragments obtained from the murine IgG1 B72.3 monoclonal antibody (MAb) by digestion with the sulfhydryl protease papain, in an attempt to determine the sites of cleavage and thus to clarify the mode of action of this enzyme on MAbs. ESI analysis of the Fab and F(ab')2 subunits indicated that the predominant site of papain cleavage occurred at C221 of the B72.3 MAb heavy chain. Reduction of the intra- and interchain disulfide bridges of these fragments by 1,4-dithiothreitol and subsequent electrospray analysis showed a loss of C221 from the C-terminal end of the Fd subunit. ESI analysis of the cleaved Fab fragment indicated that there was an apparent loss of amino acid residues from this fragment. Edman sequencing of the cleaved subunit revealed an intact light chain and the loss of QVQ from the N-terminal of the Fd subunit. Reduction of this subunit gave a Fd fragment approximately 32 Da greater than the predicted mass, which we have attributed to oxidation of the heavy chain methionine residues (M81 and M136). Removal of the carbohydrate portion from the Fc fragment by N-glycosidase F indicated that papain cleavage had occurred at C223 of the B72.3 MAb heavy chain. In addition, it was observed that the C-terminal lysine residue (K438) was absent from the deglycosylated Fc fragment, presumably due to carboxypeptidase B activity that occurs during the in vivo production of the B72.3 MAb in murine hosts. These data clearly illustrate the power of ESI-MS for determining small changes in mass on large proteins as well as providing a rapid and sensitive technique for assessing MAb fragments prior to use in radioimaging or radiotherapy.

Amino Acid Sequence↗
PubMed1997

Gamma/delta T-cell hepatosplenic lymphoma: review of the literature, diagnosis by flow cytometry and concomitant autoimmune hemolytic anemia.

Hepatosplenic gamma/delta T-cell lymphoma is recognized as a subset of peripheral T-cell lymphoma in the REAL classification. Histologically these tumors are characterized by a mixture of small to medium-sized atypical lymphocytes. To date, approximately 15 cases of hepatosplenic gamma delta T-cell lymphoma have been reported. Affected individuals are usually young adults with a median age of 34 years. Patients commonly present with B symptoms and hepatosplenomegaly, but an absence of lymphadenopathy. The disease follows an aggressive course with median survival of 12-14 months and poor response to combination chemotherapy agents. Occasionally, the occurrence of frank blast transformation constitutes a terminal event for the patient. Although cytopenias are relatively common, nonimmune hemolytic anemia has been reported in one patient only. This is the first report of autoimmune hemolytic anemia associated with hepatosplenic gamma delta T-cell lymphoma.

Adult↗
PubMed1997

Radiolabeling antibodies with holmium-166.

We report the preliminary results from radiolabeling of a chelate-conjugated antibody with 166Ho produced from the beta(-)-decay of 166Dy. Ho-166 was separated from mg quantities of Dy target by reverse phase ion-exchange chromatography employing a cation exchange HPLC column and 0.085 M alpha-HIBA at pH = 4.3 as eluent. Evaporation to dryness of 166Ho fraction (up to 25 mL) and thermal decomposition of alpha-HIBA yielded 166Ho in a dry state which was then solubilized in 0.5 mL of 0.1 M HCl. Subsequent radiolabeling of CHX-B-DTPA conjugated 135-14 monoclonal antibodies with purified 166 Ho was readily achieved with approximately 80% efficiency and with a specific activity of 3-4 mCi of 166Ho per mg of protein. 166Ho-antibody conjugates are stable with regards to transferrin challenge for a period of 50 h. Further, it was shown that any Fe3+ ions present in alpha-HIBA as an impurity interfere with the labeling.

Antibodies, Monoclonal↗
PubMed1996

Characterization of a new hereditary thrombopathy in a closed colony of Wistar rats.

A new hereditary thrombopathy has been identified in a closed colony of Wistar rats. A simple and reproducible cuticle bleeding time test was developed as a rapid screening procedure for the bleeding diathesis. Affected animals exhibit markedly prolonged bleeding times and complete absence of platelet aggregation either with adenosine diphosphate (ADP) or with thrombin. Inheritance data suggest an autosomal dominant inheritance pattern with variable penetrance. Coagulation tests, platelet counts, plasma von Willebrand factor (vWF) activity, and clot retraction are within normal limits in thrombopathic animals. GPIb-dependent botrocetin-induced platelet agglutination was present in washed thrombopathic rat platelets. No discernible abnormality of intraplatelet organelles or granules was seen by transmission electron microscopy of thrombopathic platelets. A qualitative morphologic assessment of intraplatelet fibrinogen in thrombopathic rat platelets showed no discernible difference as compared with control rat platelets. Thrombopathic rat platelets exhibit decreased glycoprotein IIb/IIIa (GPIIb/IIIa) antigen by flow cytometric analysis and markedly decreased iodine 125-labeled fibrinogen binding to platelet GPIIb/IIIa after ADP activation. This rat colony demonstrates a unique thrombopathy, distinct from previously described animal thrombopathies, with some characteristics of variant Glanzmann's thrombasthenia. This animal model may provide further insight into the regulatory mechanisms and pathophysiology of platelet GPIIb/IIIa.

Animals↗
PubMed1995

Preservation of hemostatic and structural properties of rehydrated lyophilized platelets: potential for long-term storage of dried platelets for transfusion.

Currently, therapeutic platelet concentrates can be stored for only 5 days. We have developed a procedure that permits long-term storage of fixed and lyophilized platelets that retain hemostatic properties after rehydration. These rehydrated lyophilized platelets (RL platelets) restore hemostasis in thrombocytopenic rats and become incorporated in the hemostatic plug of bleeding time wounds of normal dogs as well as von Willebrand disease dogs with partially replenished plasma von Willebrand factor. Ultrastructurally, these platelets are well preserved and are comparable to control normal washed platelets. Flow cytometry analysis shows that RL platelets react with antibodies to the major surface receptors, glycoprotein (GP)Ib and GPIIb/IIIa. These receptors are involved in platelet agglutination, aggregation, and adhesion. In vitro functional tests document the ability of RL platelets to adhere to denuded subendothelium and to spread on a foreign surface. Circulating RL platelets participated in carotid arterial thrombus formation induced in normal canine subjects. The participation of RL platelets in these vital hemostatic properties suggests that with further development they could become a stable platelet product for transfusion.

Animals↗
PubMed1995

Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein.

We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironomus tentans. Bacterially synthesized rCAS was purified to near homogeneity and characterized by several biochemical and biophysical methods including amino-terminal sequencing, amino acid compositional analysis, sedimentation equilibrium ultracentrifugation, and mass spectrometry. Complementing these techniques with quantitative sulfhydryl assays, we discovered that the four cysteines present in rCAS form two intramolecular disulfide bonds. Mapping studies revealed that the disulfide bonds are heterogeneous. When reduced and denatured rCAS was allowed to refold and its disulfide bonding state monitored, it again adopted a conformation with two intramolecular disulfide bonds. The inherent ability of rCAS to quantitatively form two intramolecular disulfide bonds may reflect a previously unknown feature of the in vivo silk proteins from which it is derived.

Amino Acid Sequence↗
PubMed1995

[166Dy]dysprosium/[166Ho]holmium in vivo generator.

A novel approach for the delivery of 166Ho (t1/2 = 26.6 h) to tissue is via the in vivo decay of its 81.5 h parent, 166Dy-an in vivo generator system. A critical question for the in vivo 166Dy/166Ho generator system is whether translocation of the daughter nucleus occurs. The in vitro and in vivo integrity of the [166Dy]Dy/166Ho-DTPA complex was investigated and results indicated that no translocation of the daughter nucleus occurs subsequent to beta- decay of 166Dy. Biodistribution studies of [166Dy]Dy-DTPA showed that the ratio of 166Dy/166Ho in bone remains constant (+/- 7%) over a 20 h period, indicating no significant in vivo loss of 166Ho from the complex. Increasing the in vivo residence time of [166Dy]Dy-DTPA complex attached to HSA gave similar results.

Animals↗
PubMed1993

GABAergic cells and signals appear together in the early post-mitotic period of telencephalic and striatal development.

Single cell suspensions derived from embryonic telencephala taken from embryos of gestational day 13 (E13) as well as rat striatal tissue from E14, 15 and 17 were prepared by tissue digestion with papain. Cell suspensions were analyzed by flow cytometry or plated onto poly-D-lysine-coated culture dishes for either nuclear staining or immunocytochemistry. Experiments on functional Na+ channels and GABAA receptor expression were carried out using a fluorescence-activated cell sorter (FACS) and a negatively charged fluorescent indicator dye (oxonol). FACS analysis of embryonic cell suspensions at E13-17 consistently revealed one major subpopulation accounting for 85-90% of the events and one minor subpopulation (10-15% of the total). When sorted, the major subpopulation consisted of phase-bright cells of 5-7 microns diameter some of which had neurites. The minor population consisted of phase-dark cells and resealed membranes of 0.5-4 microns diameter as well as debris. Almost all the cells obtained in the high FALS (forward-angle light scatter) subpopulation at E17 expressed 200-kDa neurofilament and tetanus toxin antigens while the small diameter cells seldom expressed tetanus toxin and particles never did. A small number of GABA-containing neurons were detected in the telencephalon at E13 (3%) and in the developing striatum at E14 (6%). All of the GABA-containing neurons expressed neurofilament. In the embryonic rat striatum, nanomolar concentrations of muscimol (GABAA agonist) induced depolarizing responses. A small number of cells in the high FALS subpopulation were responsive to muscimol starting at embryonic day 14, and the number of responsive cells increased at E15.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridine Orange↗
PubMed1993

The roles of von Willebrand factor and factor VIII in arterial thrombosis: studies in canine von Willebrand disease and hemophilia A.

We have studied the roles of von Willebrand factor (vWF) and factor VIII in arterial thrombosis in four canine phenotypes: normal (n = 6), hemophilia A (n = 11), von Willebrand disease (vWD) (n = 9), and hemophilia A/vWD (n = 1). vWF activity was determined by botrocetin-induced agglutination of fixed human platelets and vWF antigen (vWF:Ag) by Laurell electroimmunoassay and crossed immunoelectrophoresis. Plasma from normal dogs and those with hemophilia A had vWF activity, vWF:Ag, and a full range of vWF:Ag multimers on gel electrophoresis equivalent to normal canine plasma pool. Platelet cytosol contents were isolated by freezing and thawing, triton X-100 solubilization, or sonication of washed platelets with and without protease inhibitors and inhibitors of platelet activation. Washed platelets were also stimulated with calcium ionophore and MgCl2. There was no measurable vWF activity or vWF:Ag in platelet lysates or releasates in any dog regardless of phenotype. All dogs were studied using a standard arterial stenosis and injury procedure to induce arterial thrombosis. Thromboses were detected by cyclic reductions in Doppler blood flow velocity. Vessels were examined by light and scanning electron microscopy. Thrombosis developed in the arteries of normal (9 of 10) and hemophilia A dogs (16 of 16) but in none of the vWD dogs (0 of 10). Infusion of canine vWF cryoprecipitate into vWD dogs markedly shortened bleeding time but did not support thrombosis as seen in dogs with vWF in the plasma and subendothelium. Thrombosis, then, fails to occur when vWF is absent from the plasma and subendothelial compartments or present only in the plasma compartment. These data are consistent with the hypothesis that vWF in the plasma and subendothelium supports thrombosis. Neither plasma FVIII nor platelet vWF is essential for thrombosis in this model.

Animals↗
PubMed1993

Two functionally different glutamate receptors of the kainate subtype in embryonic rat mesencephalic cells.

The amino acid glutamate is a widespread excitatory neurotransmitter in the brain. It activates cation-selective channels expressed by nearly every neuron and by glial cells; also various second messenger cascades. Little is known about the ontogeny of glutamate neurotransmission during neurogenesis. We have analyzed the development and differentiation of excitatory amino acid responses and Na+ channels in cells dissociated from embryonic rat ventral mesencephalon and striatum as well as cortex and cerebellum using fluorescent voltage-sensitive oxonol dyes and flow cytometry. Analysis of fluorescence distribution revealed complex profiles under resting conditions which changed in a characteristic manner over the period studied (Embryonic (E) Days 12-20). The response to the Na+ channel agonist veratridine appeared at E12/13 in the mesencephalon. At E13 L-glutamate and kainate evoked changes in membrane potential interpreted as cellular hyperpolarization. At E15 some cells still responded by hyperpolarizing but an equal number began to depolarize. By E18 most cells depolarized. Both hyper- and depolarizations were eliminated by a specific antagonist at kainate receptors (6-cyano-7-nitroquinoxyline-2,3-dione) and by resuspending the cells in Na(+)-free medium. Both responses exhibited a concentration dependency with higher doses evoking stronger effects. In contrast, there was little effect of veratridine in the striatum at E15-E16, and the response to kainate or L-glutamate was predominantly depolarizing during the same embryonic period, with little or no effect until E18. These data show that in the developing CNS, sodium channel responses as well as excitatory aminoacid neurotransmitter responses first become functional in the mesencephalon and subsequently in the striatum, thus suggesting an anatomical gradient of expression. Our results also show that glutamate receptor-coupled functions vary with embryonic age and with regional distribution, suggesting possible roles of glutamate in early CNS embryogenesis, as morphogens or modulators of synaptic plasticity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1990

Electrical and chemical excitability appear one week before birth in the embryonic rat spinal cord.

Embryonic rat spinal cord cells were acutely dissociated with the enzyme papain, stained with a voltage-sensitive oxonol dye and incubated with various pharmacological agents. Changes in the fluorescence intensity and, by inference, membrane potential of the cells were analyzed in a flow cytometer. Veratridine caused depolarization of the cells in a TTX-sensitive manner from as early as embryonic day 13. Depolarizing responses to muscimol and kainate appeared slightly later, at embryonic days 14 and 15, and were blocked by the antagonists bicuculline and CNQX, respectively. Responses to veratridine and kainate did not occur in sodium-free medium. The emergence of these excitable membrane properties coincides with postmitotic differentiation and synaptic development in the embryonic spinal cord.

Animals↗
PubMed1989

Role of botrocetin in platelet agglutination: formation of an activated complex of botrocetin and von Willebrand factor.

Botrocetin (venom coagglutinin) induces binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (GPIb), resulting in platelet agglutination. A mechanism whereby botrocetin causes vWF to change to an active platelet-agglutinating form is proposed. Incubation of native vWF with botrocetin yielded an increasingly active vWF with slower migration in two-dimensional immunoelectrophoresis but with no apparent change in vWF multimer pattern. The "activated" vWF eluted mainly in the void volume (Vo) (Bio-Gel A-15m column chromatography). Botrocetin eluted in the included volume (Vi). Vo peaks appeared to contain a vWF-botrocetin complex, based on bioassays and immunoassays. 125I-Botrocetin mixed with vWF eluted in two peaks: in the Vo, coincident with active vWF, and in the Vi. With von Willebrand disease (vWD) plasma lacking vWF, 125I-Botrocetin eluted in the Vi only. It did not bind to platelets without vWF. In aggregometric studies, antibodies (Ab) against botrocetin, vWF, and GPIb prevented botrocetin-induced platelet agglutination and caused dissolution of preformed platelet agglutinates. Immunostaining of aggregates with antibotrocetin Ab revealed a positive reaction. Botrocetin appears to act in a two-step manner, first binding with vWF to form a complex, which then binds to GPIb to cause agglutination. All three components, vWF, botrocetin, and GPIb, appear to be required for maintenance of stable platelet agglutinates.

Animals↗
PubMed1987

Effects of continuous intravenous infusion of an ovine placental extract enriched in placental lactogen on plasma hormones, metabolites and metabolite biokinetics in non-pregnant sheep.

Continuous intravenous infusions of saline or of a placental extract containing ovine placental lactogen were given to three non-pregnant, non-lactating ewes over periods of 36 h, 1 week apart. During saline infusion no placental lactogen was detected in jugular vein plasma. but infusion of the placental extract raised the placental lactogen concentration from undetectable to 40-50 micrograms/l, similar to concentrations in ewes with one fetus on day 90 of pregnancy. By comparison with the saline control period, infusion of the placental extract consistently increased both plasma concentrations and irreversible loss of nonesterified fatty acids. Plasma concentrations of glucose and urea, but not irreversible loss of these metabolites, were consistently increased. Although the placental extract was not subjected to extensive purification, it was enriched in placental lactogen and contained no detectable contamination with insulin, prolactin or growth hormone. The results are suggestive of a role for placental lactogen in modifying metabolism and acting during pregnancy to provide nutrients for fetal metabolism.

Animals↗