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J L Halpern

Publications and source records attributed to J L Halpern.

At least 19 recordsLinked to original sources
PubMed1996

Clostridial neurotoxins and substrate proteolysis in intact neurons: botulinum neurotoxin C acts on synaptosomal-associated protein of 25 kDa.

Clostridial neurotoxins are zinc endopeptidases that block neurotransmission and have been shown to cleave, in vitro, specific proteins involved in synaptic vesicle docking and/or fusion. We have used immunohistochemistry and immunoblotting to demonstrate alterations in toxin substrates in intact neurons under conditions of toxin-induced blockade of neurotransmitter release. Vesicle-associated membrane protein, which colocalizes with synaptophysin, is not detectable in tetanus toxin-blocked cultures. Syntaxin, also concentrated in synaptic sites, is cleaved by botulinum neurotoxin C. Similarly, the carboxyl terminus of the synaptosomal-associated protein of 25 kDa (SNAP-25) is not detectable in botulinum neurotoxin A-treated cultures. Unexpectedly, tetanus toxin exposure causes an increase in SNAP-25 immunofluorescence, reflecting increased accessibility of antibodies to antigenic sites rather than increased expression of the protein. Furthermore, botulinum neurotoxin C causes a marked loss of the carboxyl terminus of SNAP-25 when the toxin is added to living cultures, whereas it has no action on SNAP-25 in vitro preparations. This study is the first to demonstrate in functioning neurons that the physiologic response to these toxins is correlated with the proteolysis of their respective substrates. Furthermore, the data demonstrate that botulinum neurotoxin C, in addition to cleaving syntaxin, exerts a secondary effect on SNAP-25.

Animals↗
PubMed1996

Rating long-term care facilities on pressure ulcer development: importance of case-mix adjustment.

OBJECTIVE: To determine the importance of case-mix adjustment in interpreting differences in rates of pressure ulcer development in Department of Veterans Affairs long- term care facilities. DESIGN: A sample assembled from the Patient Assessment File, a Veterans Affairs administrative database, was used to derive predictors of pressure ulcer development; the resulting model was validated in a separate sample. Facility-level rates of pressure ulcer development, both unadjusted and adjusted for case mix using the predictive model, were compared. SETTING: Department of Veterans Affairs long-term care facilities. PATIENTS: The derivation sample consisted of 31 150 intermediate medicine and nursing home residents who were initially free of pressure ulcers and were institutionalized between October 1991 and April 1993. The validation sample consisted of 17 946 residents institutionalized from April 1993 to October 1993. MEASUREMENT: Development of a stage 2 or greater pressure ulcer. RESULTS: 11 factors predicted pressure ulcer development. Validated performance properties of the resulting model were good. Model-predicted rates of pressure ulcer development at individual long-term care facilities varied from 1.9% to 6.3%, and observed rates ranged from 0% to 10.9%. Case-mix-adjusted rates and ranks of facilities differed considerably from unadjusted ratings. For example, among five facilities that were identified as high outliers on the basis of unadjusted rates, two remained as outliers after adjustment for case mix. CONCLUSIONS: Long-term care facilities differ in case mix. Adjustments for case mix result in different judgments about facility performance and should be used when facility incidence rates are compared.

Aged↗
PubMed1994

Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells.

The light chain of tetanus toxin is a zinc endoprotease that inhibits neurotransmitter release by selective proteolysis of the synaptic vesicle-associated protein synaptobrevin/vesicle-associated membrane protein. Cellubrevin is a homologue of synaptobrevin that is found in most cell types and is also a substrate for tetanus toxin. The lack of receptors for tetanus toxin on most cell types has made studies of tetanus toxin action in non-neuronal cells difficult. To characterize tetanus toxin effects in non-neuronal cells, a fusion protein consisting of the 254 amino-terminal amino acids of lethal factor (LF) of anthrax toxin and tetanus toxin light chain (LC) was prepared. This protein (LF-LC) inhibited evoked glycine release from primary spinal cord neurons at concentrations between 1.0 and 100 ng/ml. LF-LC was cytotoxic to RAW 264.7, ANA-1 cells (mouse macrophage cell lines), and Chinese hamster ovary cells in a dose-dependent manner. These effects required the presence of protective antigen, the receptor binding component of anthrax toxin. In contrast, LF-LC was not cytotoxic to RBL-2H3, Vero, or mouse hybridoma cell lines. Mutagenesis of conserved amino acids (His237 and Glu234) in the zinc-binding motif of LC resulted in fusion proteins having no biological activity. LF-LC did not inhibit regulated secretion of serotonin in RBL-2H3 cells or constitutive secretion in any non-neuronal cell lines as measured in several different assays. We suggest that the cytotoxic effects of LF-LC result from inhibition of a specific intracellular membrane fusion event mediated by cellubrevin.

Animals↗
PubMed1993

Characterization of the receptor-binding domain of tetanus toxin.

The carboxyl-terminal half of the heavy chain of tetanus toxin (Hc) contains the domain required for binding to purified gangliosides and neuronal cells. The structural requirements for the interaction of Hc with receptor were studied by generating mutants of Hc with deletions at either the carboxyl or amino terminus and characterizing their binding. A deletion of 10 or more amino acids from the carboxyl terminus resulted in a major loss of Hc binding to purified gangliosides and spinal cord neuronal cells, whereas a deletion of the carboxyl-terminal 5 amino acids did not affect binding. The removal of up to 263 amino acids from the amino terminus did not inhibit binding. Each of the truncated proteins was much more sensitive to trypsin than was full-length Hc, suggesting an alteration in conformation. The receptor binding activity of Hc was not retained in a peptide corresponding to the carboxyl-terminal 20 amino acids. These data suggest that the carboxyl-terminal region of Hc is important for maintaining a conformation necessary for binding to receptor.

Amino Acid Sequence↗
PubMed1992

A simplified method for the preparation of tetanus toxin binding fragment for neurobiology.

The non-toxic binding fragment of tetanus toxin (fragment C) binds avidly to neural tissue and has a growing number of neurobiological uses. Its current utility is limited by both its high commercial cost and the complex procedure for its preparation requiring highly purified tetanus toxin. We have developed a short procedure which prepares fragments of tetanus toxin from crude C. tetani extracts. The resultant proteins are atoxic with molecular sizes and immunological properties closely resembling fragment C. These proteins undergo retrograde axonal and apparent transneuronal transport in a fashion similar to fragment C.

Animals↗
PubMed1990

Effect of tetanus toxin on oxytocin and vasopressin release from nerve endings of the neurohypophysis.

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.

Animals↗
PubMed1990

Cloning and expression of functional fragment C of tetanus toxin.

A segment of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by using the polymerase chain reaction. This fragment was cloned into expression vector pTTQ8, under the control of the tac promoter. Expression of this plasmid in Escherichia coli resulted in the production of a protein consisting of 8 amino acids of the vector fused to the C-terminal 460 amino acids of tetanus toxin. This protein (rFragment C) was recognized by an antipeptide antibody specific for fragment C in an enzyme-linked immunosorbent assay and on immunoblots. rFragment C could be purified significantly in one step by immunoaffinity chromatography. Immunization of mice with rFragment C resulted in the production of antibodies that were able to protect the mice against a challenge with tetanus toxin. rFragment C bound to ganglioside GT1b and to neuronal cells in a manner indistinguishable from that of fragment C obtained by papain cleavage of tetanus toxin. For many applications, rFragment C appears to be a suitable alternative to tetanus toxin or toxin-derived fragment C.

Animals↗
PubMed1990

Immunological characterization of guanine nucleotide-binding proteins: effects of a monoclonal antibody against the gamma subunit of transducin on guanine nucleotide-binding protein-receptor interactions.

Guanine nucleotide-binding proteins (G proteins) transduce signals from agonist- and light-sensitive receptors. In the visual excitation system, the photon receptor rhodopsin is coupled to the G protein Gt (transducin). Gt is composed of alpha, beta, and gamma subunits; the alpha subunit binds guanine nucleotide, whereas the beta and gamma subunits, which are tightly associated, appear to facilitate interaction of alpha with receptor and pertussis toxin-catalyzed ADP-ribosylation of alpha. To study the function of transducin, monoclonal antibodies were developed against the purified protein. Monoclonal antibody 2H3 reacted with Gt gamma but not G gamma from bovine brain or rabbit liver. In the absence of photolyzed rhodopsin, both intact 2H3 and Fab fragments of 2H3 were able to inhibit completely, in a concentration-dependent manner, ADP-ribosylation of transducin by pertussis toxin 2H3 had no effect on ADP-ribosylation in the presence of photolyzed rhodopsin. The GTPase activity of transducin, which is dependent on rhodopsin, was inhibited only 50% by 2H3. These data are consistent with the hypotheses that an epitope recognized by 2H3 may be important in the formation of the alpha beta gamma complex or that interaction of 2H3 with gamma may alter conformation of the latter and, thereby, inhibit complex formation. Further, reactions of gamma with 2H3 appear to be prevented by interaction with rhodopsin, suggesting that its interaction either shields or alters the epitope recognized by 2H3.

Animals↗
PubMed1989

Isolation and immunological properties of adenosine kinase.

Bovine liver adenosine kinase is a 43 kDa protein that catalyzes the transfer of phosphate from GTP or ATP to adenosine. Its immunological properties were compared to other GTP-binding proteins of approximately 40 kDa, in particular those involved in signal transduction, such as Gs and Gi, the stimulatory and inhibitory regulatory proteins of adenylyl cyclase, Gt, from the visual excitation system, and Go, a similar protein of unknown function. Antibodies elicited in rabbits against adenosine kinase did not significantly cross-react with other guanyl nucleotide-binding proteins. Antibodies against the other GTP-binding proteins did not react with adenosine kinase. Thus these GTP-binding proteins do not exhibit immunological cross-reactivity.

Adenosine Kinase↗
PubMed1989

Monoclonal antibodies to Pneumocystis carinii: identification of specific antigens and characterization of antigenic differences between rat and human isolates.

To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii reacted with human P. carinii, and none of four monoclonal antibodies to human P. carinii reacted with rat P. carinii. Two antibodies to human P. carinii reacted by immunofluorescence with only one human P. carinii isolate. Immunoblot studies identified major antigens of rat P. carinii with molecular masses of 40,000-100,000 daltons and of human P. carinii with molecular masses of 22,000-95,000 daltons. These studies document the existence of antigenic differences between rat and human P. carinii and are consistent with the suggestion that individual isolates of human P. carinii are also antigenically different. Further studies with these antibodies should increase understanding of the antigenic nature of P. carinii and of the interaction of P. carinii with its host.

Animals↗
PubMed1989

Sequence homology between tetanus and botulinum toxins detected by an antipeptide antibody.

The extent of immunological similarity between tetanus toxin and botulinum toxins A, B, C1, and E was studied by using 10 antibodies produced against synthetic peptides representing different sequences of tetanus toxin, mouse antitetanus serum, and human Tetanus Immune Globulin. Antibodies produced against the synthetic peptides recognized tetanus toxin in an enzyme-linked immunosorbent assay and on Western blots (immunoblots) but did not appear to recognize the native protein. One of the antitetanus peptide antibodies, which was produced against a peptide from the amino terminal, cross-reacted with three of the four botulinum toxins on immunoblots. This antibody, 1, reacted strongly with botulinum toxins B and C1 and weakly with E but did not recognize type A toxin. None of the other peptide antibodies cross-reacted with the botulinum toxins. Mouse antitetanus serum and human Tetanus Immune Globulin did not recognize any of the botulinum toxins on immunoblots. The amino-terminal region of the light chain of tetanus toxin and botulinum toxin types A, B, C1, and E are known to have sequence homology. Our data demonstrate that for tetanus toxin and botulinum toxin types B, C1, and E this region also has immunological homology. Type A, which has the least amount of homology with tetanus toxin in this region, does not share this immunological homology. These data also suggest that although the native structures of tetanus and botulinum toxins have relatively few common immunological determinants, the two toxins may contain short stretches of identical or very similar amino acid sequences.

Amino Acid Sequence↗
PubMed1988

Identification of antigens and antibodies specific for Pneumocystis carinii.

To increase understanding of Pneumocystis carinii and its interaction with its hosts, Ag specific for rodent and human P. carinii were identified by the immunoblot method after PAGE of P. carinii organism extracts. The m.w. of the major Ag of rat P. carinii were 45,000, 110,000, and a broad band of 49,000 to 64,000, and of human P. carinii were 22,000, 24,000, and a broad band of 35,000 to 45,000 daltons. Human and rat pneumocystis were not antigenically identical. Specific antibodies against rat P. carinii Ag were found in 18 of 79 rats by the immunoblot method. Specific antibodies against human P. carinii Ag were found in 32 of 33 adult human sera, but in only 1 of 8 sera from infants and children. Specific antibodies were found in sera of 13 of the 14 adults with no history of P. carinii pneumonia, and all 19 patients with recently diagnosed P. carinii pneumonia, including 9 patients with P. carinii pneumonia associated with AIDS. The results of this study support previous suggestions that a large proportion of adults have been exposed to P. carinii and provide a basis for the further investigations of host-P. carinii interactions.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Mechanism of enhanced sensitivity to bradykinin in pertussis toxin-treated fibroblasts: toxin increases bradykinin-stimulated prostaglandin formation.

Exposure of animals to pertussis toxin results in increased sensitivity to agents such as bradykinin. To elucidate the molecular mechanisms underlying the effects of toxin, bradykinin responsiveness was examined in control and intoxicated human fibroblasts. Exposure of fibroblasts to toxin resulted in a loss of inhibitory agonist action on adenylate cyclase, elevation of basal cAMP, and ADP-ribosylation of a 41-kDa protein, which was identified as Gi alpha, a component of adenylate cyclase, by its pattern of immuno-cross-reactivity with a family of antibodies to guanyl nucleotide-binding proteins, which are pertussis toxin substrates, and by the presence of an mRNA species with characteristics of a form of Gi alpha. Bradykinin increased prostaglandin accumulation to a greater extent in toxin-treated than in control fibroblasts. Agents such as cholera toxin, which elevated cAMP, also increased bradykinin-induced prostaglandin production. These data are consistent with the hypothesis that the enhanced sensitivity to bradykinin after pertussis toxin treatment results from modification of Gi alpha and increased cAMP, leading to enhanced formation of prostaglandins in response to bradykinin.

Adenosine Diphosphate Ribose↗
PubMed1987

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha.

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Production of antibodies against rhodopsin after immunization with beta gamma-subunits of transducin: evidence for interaction of beta gamma-subunits of guanosine 5'-triphosphate binding proteins with receptor.

The light-detecting system of retinal rod outer segments is regulated by a guanyl nucleotide binding (G) protein, transducin, which is composed of alpha-, beta-, and gamma-subunits. Transducin couples rhodopsin to the intracellular effector enzyme, a cGMP phosphodiesterase. The beta gamma complex (T beta gamma) is required for the alpha-subunit (T alpha) to interact effectively with the photon receptor rhodopsin. It is not clear, however, whether T beta gamma binds directly to rhodopsin or promotes T alpha binding to rhodopsin only by binding to T alpha. We have found that serum from rabbits immunized with T beta gamma contained a population of antibodies that were reactive against rhodopsin. These antibodies could be separated from T beta gamma antibodies by absorbing the latter on immobilized transducin. Binding of purified rhodopsin antibodies was inhibited by T beta gamma, suggesting that the rhodopsin antibodies and T beta gamma bound to the same site on rhodopsin. We propose that the rhodopsin antibodies act both as antiidiotypic antibodies against the idiotypic T beta gamma antibodies and as antibodies against rhodopsin. This hypothesis is consistent with the conclusion that T beta gamma interacts directly with the receptor. It is probable that in an analogous way, G beta gamma interacts directly with receptors of the adenylate cyclase system.

Animals↗
PubMed1986

Structural and functional characterization of guanyl nucleotide-binding proteins using monoclonal antibodies to the alpha-subunit of transducin.

Transducin, the GTP-binding protein of the retinal light-sensitive phosphodiesterase system, and Gs and Gi, regulatory proteins of the hormone-sensitive adenylate cyclase, are members of a family of guanyl nucleotide-binding proteins termed G proteins that are important in signal transduction. To probe relationships within this family of G proteins, monoclonal antibodies were prepared against the alpha-subunit of bovine transducin (T alpha). Three of four monoclonal antibodies were specific for T alpha and did not cross-react with other G proteins. One, MAB1, cross-reacted strongly with the alpha-subunit of Gi (Gi alpha) purified from rabbit liver and, to a lesser extent, with the alpha-subunit of Go (Go alpha) purified from bovine brain and the proto-oncogene product H-ras p21. All four monoclonal antibodies recognized epitopes on a 23-kDa tryptic peptide fragment of T alpha which is derived from the N-proximal region. The three monoclonal antibodies that recognized only T alpha inhibited rhodopsin-stimulated GTP binding and hydrolysis by transducin, whereas MAB1 had no significant effect in these assays. These studies demonstrate that, within the 23-kDa tryptic peptide of T alpha, there is a domain(s) unique to T alpha that is involved in GTP binding and hydrolysis and another domain which is highly conserved in T alpha and to a lesser extent in other G proteins. Prior studies have identified regions involved in nucleotide binding and hydrolysis that are homologous in all G proteins. The observations reported here are consistent with the conclusion that the G proteins may have in addition unique regions involved in these functions.

Amino Acid Sequence↗
PubMed1984

Pertussis toxin-catalyzed ADP-ribosylation: effects on the coupling of inhibitory receptors to the adenylate cyclase system.

The adenylate cyclase system consists of stimulatory and inhibitory hormone and drug receptors coupled through different GTP-binding proteins to a catalytic unit, responsible for the synthesis of cAMP from ATP. Pertussis toxin blocks the effect of inhibitory agonists on the catalytic unit by enzymatically inactivating the inhibitory GTP-binding protein (Gi). Study of the inhibitory arm of the cyclase system has been facilitated by the dissection of the overall process of hormonal inhibition of cAMP formation into a series of reactions characteristic of the individual protein components of this complex system; pertussis toxin has proven to be a useful tool with which to study these individual reactions. Exposure of cells or membranes to pertussis toxin in the presence of NAD results in ADP-ribosylation of a 41,000 Da subunit of Gi. ADP-ribosylation of Gi has a number of effects on the overall and partial reactions of the cyclase system, including a loss of a) hormonal inhibition of cAMP formation, b) hormonal stimulation of GTPase and c) agonist-induced release of membrane-bound guanyl nucleotides. In addition, in toxin-treated membranes, the affinity of inhibitory receptors for agonist but not antagonist is decreased with no significant change in receptor number.

Adenosine Diphosphate↗