PubMed Health⌕ Search

Biomedical subjects

J E Maggio

Publications and source records attributed to J E Maggio.

At least 37 records · Page 2Linked to original sources
PubMed1996

Intra-arterial infusion of [125I]A beta 1-40 labels amyloid deposits in the aged primate brain in vivo.

Alzheimer's disease is characterized by extracellular amyloid deposits in the brain at both vascular sites (cerebrovascular amyloid, CVA) and within the neuropil (plaques). In the present study we demonstrated that brain amyloid of aged non-human primates is efficiently detected by [125I]A beta in vitro, and assessed the detection of that amyloid in vivo by intravascular infusion of [125I]A beta. Aged squirrel monkeys (Saimiri sciureus) were anesthetized and infused intra-arterially with [125I]A beta, and sacrificed 2 h later. Analysis of the anterior frontal and temporal cortices by autoradiography demonstrated that [125I]A beta was deposited on CVA and that essentially every amyloid deposit which could be detected with thioflavin S or anti-A beta antibodies was also labeled by [125I]A beta. These experiments suggest that intravascular infusion of radiolabeled A beta can be used to detect and image amyloid deposits in the human AD brain.

Aging↗
PubMed1996

Differential expression of two isoforms of the neurokinin-1 (substance P) receptor in vivo.

Recent pharmacological and biochemical studies have suggested that there may be more than one molecular form of the neurokinin-1 receptor (NK-1), a long and short isoform differing in the length of their cytoplasmic carboxyl-terminal tails, but no definitive evidence of the existence of such NK-1 receptor isoforms in tissue has been presented. To examine whether these different isoforms are expressed in vivo we have compared the distribution of high affinity substance P (SP) binding sites (visualized by autoradiography with [125I]SP), with the distribution of the C-terminal epitope of the full length receptor (visualized with a specific antibody against the extreme C-terminal sequence). The former method labels both long and short forms of the NK-1 receptor, while the latter labels only the long form of the protein. In the rat there is a close correspondence of [125I]SP binding and NK-1 immunoreactivity in the striatum, suggesting that the long isoform predominates in this tissue. In the parotid and submaxillary gland, there are very high levels of [125I]SP binding but only low levels of NK-1 immunoreactivity, suggesting that expression of the short form predominates in these tissues. These results imply that different tissues express different ratios of the two isoforms of the NK-1 receptor. This differential expression provides the theoretical basis for tissue specific pharmacological targeting of NK-1 receptors.

Analysis of Variance↗
PubMed1996

In vitro growth of Alzheimer's disease beta-amyloid plaques displays first-order kinetics.

A salient pathological feature of Alzheimer's disease (AD) is the presence of amyloid plaques in the brains of affected patients. The plaques are predominantly composed of human beta-amyloid peptide (A beta). Although the aggregation of synthetic A beta has been extensively studied, the mechanism of AD plaque growth is poorly understood. In order to address this question, we used an in vitro model of plaque growth to determine if assembly or aggregation of A beta is required for deposition. Labeled A beta at physiological concentrations readily deposited onto both neuritic and diffuse plaques and cerebrovascular amyloid in unfixed AD brain tissue, whereas essentially no deposition was detected in tissue without performed amyloid. Using this in vitro model of plaque growth, the kinetics of A beta deposition onto plaques was examined in two independent but complementary systems. Intact sections of unfixed AD brain cortex (analyzed by autoradiographic densitometry) allowed definitive morphological analysis of the site of deposition, while homogenates of the same tissue (analyzed by radioisotope counting) allowed precise quantitation of deposition over a wide range of conditions. Essentially identical results were obtained for both systems. Growth of preexisting tissue plaques by deposition of A beta was found to follow first-order dependence on A beta concentration and exhibited a pH optimum of 7. In sharp contrast, A beta aggregation in the absence of template follows higher order kinetics and shows a pH optimum of 5. On the basis of criteria of kinetic order, pH dependence, and structure-activity relationships, we conclude that aggregation of A beta (template-independent initial nidus formation) and deposition of A beta (template-dependent subsequent plaque growth) are fundamentally distinct biochemical processes. The process of plaque growth and maturation by A beta deposition may be an important target for therapeutic intervention to block the progression of AD.

Alzheimer Disease↗
PubMed1996

Increased substance P receptor expression by blood vessels and lymphoid aggregates in Clostridium difficile-induced pseudomembranous colitis.

Pseudomembranous colitis is most often caused by toxins secreted by Clostridium difficile following bowel flora overgrowth after antibiotic use. The secretory and inflammatory effects observed in C. difficile toxin A-induced enterocolitis in the rat ileum are inhibited by CP-96,345, a substance P (SP) receptor antagonist. To determine if SP plays a role in the pathogenesis of human pseudomembranous colitis, SP receptor distribution was examined in a toxin A-positive specimen of bowel. Quantitative receptor autoradiography was used to examine SP receptors in tissue from a patient who tested positive for C. Difficile toxin. SP receptors were massively increased in small blood vessels and lymphoid aggregates in the pseudomembranous colitis bowel in comparison to control specimens. The SP binding was saturable and exhibited similar affinities for SP and CP-96,345. SP may contribute to the inflammatory response in pseudomembranous colitis via a massive increase in SP receptor antagonists may offer a novel therapeutic intervention for pseudomembranous colitis.

Adult↗
PubMed1996

Zinc-induced aggregation of human and rat beta-amyloid peptides in vitro.

The major pathological feature of Alzheimer's disease is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human beta-amyloid peptide (A beta), a 39-43-mer peptide the neurotoxicity of which is related to its aggregation state. Previous work has demonstrated that certain metals that have been implicated as risk factors for Alzheimer's disease (Al, Fe, and Zn) also cause substantial aggregation of A beta. In particular, we reported that zinc cations at concentrations of > 10(-4) M dramatically accelerate the rate of A beta aggregation at physiological peptide concentrations at 37 degrees C in vitro. In the present study, we investigate the effect of Zn2+ on aggregation of radiolabeled and unlabeled human and rat A beta over a wide range of peptide concentrations in the presence and absence of salt and blocking protein. Aggregation was assayed by centrifugation and filtration using amino acid analysis, immunoassay, and gamma-counting for quantification over a wide range of concentrations of Zn2+ and A beta above and below physiological values. The results of this study demonstrate the following: (a) Radioiodinated A beta accurately tracked unlabeled A beta, (b) zinc concentrations of at least 10(-4) M were required to induce significant aggregation of A beta, and (c) rat and human A beta species were cleared from aqueous solutions by similar concentrations of zinc. These results stand in significant quantitative disagreement (approximately 100-fold in zinc concentration) with one previous study that reported significant aggregation of A beta by < 1 microM Zn2+. Differences between the present study and the latter study from another laboratory appear to result from inappropriate reliance on optical density to measure A beta concentrations and nonspecific loss of A beta to plastic in the absence of blocking protein.

Amyloid beta-Peptides↗
PubMed1996

Brain amyloid--a physicochemical perspective.

The ability to form stable cross-beta fibrils is an intrinsic physicochemical characteristic of the human beta-amyloid peptide (A beta), which forms the brain amyloid of Alzheimer's disease (AD). The high amyloidogenicity and low solubility of this hydrophobic approximately 40-mer have been barriers to its study in the past, but the availability of synthetic peptide and new physical methods has enabled many novel approaches in recent years. Model systems for A beta aggregation (relevant to initial nidus formation) and A beta deposition (relevant to plaque growth and maturation) in vitro have allowed structure/activity relationships and kinetics to be explored quantitatively, and established that these processes are biochemically distinct. Different forms of the peptide, with different physiochemical characteristics, are found in vascular and parenchymal amyloid. Various spectroscopic methods have been used to explore the three-dimensional conformation of A beta both in solution and in solid phase, and demonstrated that the peptide adopts a different configuration in each state. A significant conformational transition is essential to the transformation of A beta from solution to fibril. These observations suggest new therapeutic targets for the treatment of AD.

Alzheimer Disease↗
PubMed1995

Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation.

In vivo somatosensory stimuli evoked the release of substance P from primary afferent neurons that terminate in the spinal cord and stimulated endocytosis of substance P receptors in rat spinal cord neurons. The distal dendrites that showed substance P receptor internalization underwent morphological reorganization, changing from a tubular structure to one characterized by swollen varicosities connected by thin segments. This internalization and dendritic structural reorganization provided a specific image of neurons activated by substance P. Thus receptor internalization can drive reversible structural changes in central nervous system neurons in vivo. Both of these processes may be involved in neuronal plasticity.

Animals↗
PubMed1995

Morphological characterization of substance P receptor-immunoreactive neurons in the rat spinal cord and trigeminal nucleus caudalis.

Although there is considerable evidence that primary afferent-derived substance P contributes to the transmission of nociceptive messages at the spinal cord level, the population of neurons that expresses the substance P receptor, and thus are likely to respond to substance P, has not been completely characterized. To address this question, we used an antibody directed against the C-terminal portion of the rat substance P receptor to examine the cellular distribution of the receptor in spinal cord neurons. In a previous study, we reported that the substance P receptor decorates almost the entire dendritic and somatic surface of a subpopulation of spinal cord neurons. In the present study we have taken advantage of this labeling pattern to identify morphologically distinct subpopulations of substance P receptor-immunoreactive neurons throughout the rostral-caudal extent of the spinal cord. We observed a dense population of fusiform substance P receptor-immunoreactive neurons in lamina I at all segmental levels. Despite having the highest concentration of substance P terminals, the substantia gelatinosa (lamina II) contained almost no substance P receptor-immunoreactive neurons. Several distinct populations of substance P receptor-immunoreactive neurons were located in laminae III-V; many of these had a large, dorsally directed dendritic arbor that traversed the substantia gelatinosa to reach the marginal layer. Extensive labeling was also found in neurons of the intermediolateral cell column. In the ventral horn, we found that labeling was associated with clusters of motoneurons, notably those in Onuf's nucleus in the sacral spinal cord. Finally, we found no evidence that primary afferent fibers express the substance P receptor. These results indicate that relatively few, but morphologically distinct, subclasses of spinal cord neurons express the substance P receptor. The majority, but not all, of these neurons are located in regions that contain neurons that respond to noxious stimulation.

Afferent Pathways↗
PubMed1995

Rapid endocytosis of a G protein-coupled receptor: substance P evoked internalization of its receptor in the rat striatum in vivo.

Studies on cultured cells have shown that agonists induce several types of G protein-coupled receptors to undergo internalization. We have investigated this phenomenon in rat striatum, using substance P (SP)-induced internalization of the SP receptor (SPR) as our model system. Within 1 min of a unilateral striatal injection of SP in the anesthetized rat, nearly 60% of the SPR-immunoreactive neurons within the injection zone display massive internalization of the SPR--i.e., 20-200 SPR+ endosomes per cell body. Within the dendrites the SPR undergoes a striking translocation from the plasma membrane to endosomes, and these dendrites also undergo a morphological reorganization, changing from a structure of rather uniform diameter to one characterized by large, swollen varicosities connected by thin fibers. In both cell bodies and dendrites the number of SPR+ endosomes returns to baseline within 60 min of SP injection. The number of neurons displaying substantial endosomal SPR internalization is dependent on the concentration of injected SP, and the SP-induced SPR internalization is inhibited by the nonpeptide neurokinin 1 receptor antagonist RP-67,580. These data demonstrate that in the central nervous system in vivo, SP induces a rapid and widespread SPR internalization in the cell bodies and dendrites and a structural reorganization of the dendrites. These results suggest that many of the observations that have been made on the internalization and recycling of G protein-coupled receptors in in vitro transfected cell systems are applicable to similar events that occur in the mammalian central nervous system in vivo.

Analysis of Variance↗
PubMed1995

Mapping peptide-binding domains of the substance P (NK-1) receptor from P388D1 cells with photolabile agonists.

The tachykinin substance P (SP) is a peptide transmitter of primary afferents. Its actions on both central and peripheral targets are mediated by a G-protein-coupled receptor of known primary structure. To identify contact sites between the undecapeptide SP and its receptor, we prepared radiolabeled photoreactive analogs of SP (H-RPKPQQFFGLM-NH2) by replacing amino acids in the peptide with p-benzoyl-L-phenylalanine (BPA). SP, BPA3-SP, and BPA8-SP bind with high affinity (Kd < 3 nM) to SP receptors on the murine cell line P388D1, triggering intracellular calcium responses. Both binding and calcium responses are blocked by the specific SP receptor antagonist CP-96345. On photolysis, radioiodinated BPA3-SP, and BPA8-SP covalently label a heterogeneously glycosylated protein of about 75 kDa; labeling is abolished by excess unlabeled SP or CP-96345. The labeled receptors were digested with V8 protease and/or trypsin, and the resulting fragments were analyzed by electrophoresis, high pressure liquid chromatography, and chemical or enzymatic modification. BPA3-SP and BPA8-SP photo-incorporate into different regions of the murine SP receptor. The results establish that the third and the eighth positions of SP, respectively, interact with the NH2-terminal extracellular tail (residues 1-21) and second extracellular loop (residues 173-183) of the SP receptor. A model for the agonist peptide-binding sites of the SP receptor is proposed based on photoaffinity labeling and mutagenesis studies.

Affinity Labels↗
PubMed1995

Differential expression of substance P receptors in patients with Crohn's disease and ulcerative colitis.

BACKGROUND & AIMS: Although clinical and pathological differences exist between Crohn's disease (CD) and ulcerative colitis (UC), distinguishing features are often absent, making diagnosis and treatment problematic. This study evaluated the differences in the expression of substance P (SP) receptors in patients with CD or UC. METHODS: Tissue samples from patients with inflammatory bowel disease or control patients were obtained at surgery, processed for 125I-SP binding, and analyzed by quantitative autoradiography. RESULTS: Patients with CD showed a massive increase in SP receptors in lymphoid aggregates, small blood vessels, and enteric neurons of the small and large bowel relative to controls. Six of 16 CD specimens had no pathological evidence of CD yet continued to express high concentrations of SP receptors. Pathologically positive patients with UC showed high concentrations of SP receptors on colonic lymphoid aggregates and small blood vessels but not enteric neurons. No increased SP binding was evident in clinically and pathologically quiescent UC colons and normal UC ileostomy samples. CONCLUSIONS: The increased expression of SP receptors on the enteric neurons of patients with CD distinguishes CD from UC. The persistent increased SP binding in pathologically normal CD tissue may indicate a subclinical disease state. SP receptor expression may have important diagnostic, etiologic, and therapeutic usefulness in inflammatory bowel disease.

Adult↗
PubMed1995

Simultaneous extraction of total RNA and peptides from tissues: application to tachykinins.

Methods for extraction and isolation of intact RNA are often laborious, time consuming, and preclude the direct analysis of peptides. Similarly, the conditions for extraction and isolation of peptides are unsuitable for the isolation of intact RNA. Thus, to study changes in the levels of neuropeptides and gene expression of the corresponding mRNAs, separate procedures are required. A simple and rapid method for the simultaneous extraction of RNA and peptides from tissues is described. RNA and peptides are extracted with guanidinium isothiocyanate, followed by delipidation, and peptides are isolated by a simple solid-phase extraction procedure. RNA is isolated by differentially partitioning DNA into an organic phase, followed by precipitation with ethanol. The RNA and peptides isolated by this method are of high yield and quality. Furthermore, this method for RNA isolation is successful and efficient, even with tissues that proved recalcitrant with other procedures, and allows the simultaneous processing of multiple samples. We describe the successful application of this procedure for measuring tachykinins and the corresponding preprotachykinin A mRNA from tissues. Extraction of neuropeptide K, a 36-mer tachykinin, was dramatically more efficient with the present method than other methods in common use.

Animals↗
PubMed1995

Local anesthetics inhibit substance P binding and evoked increases in intracellular Ca2+.

BACKGROUND: During spinal and epidural anesthesia, local anesthetics reach concentrations in cerebrospinal fluid and spinal cord tissues at which their actions may extend beyond the classic blockade of sodium channels. This study examines the effects of several clinical and experimental local anesthetics on the binding and actions of a peptide neurotransmitter, substance P, known to be important in nociceptive transmission in the dorsal horn. METHODS: The binding of radiolabeled (Bolton-Hunter modified) substance P was studied in chick brain membranes in the presence of local anesthetics. The increase in intracellular calcium [Ca2+]in evoked by substance P was measured by the fluorescent indicator fura-2 loaded in a murine cell line expressing substance P (NK1) receptors. Cells were preincubated with bupivacaine before and during the transient addition of substance P. RESULTS: Both substance P binding and Ca2+ increase were inhibited half-maximally by approximately 1 mM bupivacaine at pH 7.5, whereas tetracaine, lidocaine, and benzocaine were slightly less potent at inhibiting binding. Concentration-dependent substance P-binding studies showed that bupivacaine's inhibition was not competitive. Inhibition of substance P binding by bupivacaine increased with increasing pH, but the protonated species appears to have some inhibitory activity, and quaternary lidocaine also inhibited binding. There was no stereoselectively to the binding inhibition. CONCLUSIONS: Because millimolar concentrations of local anesthetics are within the range measured in spinal cord during intrathecal and epidural procedures, these results are consistent with a direct action of local anesthetics on tachykinin-mediated neurotransmission during regional anesthesia.

Anesthetics, Local↗
PubMed1995

Beta 2-adrenergic receptors are expressed by glia in vivo in the normal and injured central nervous system in the rat, rabbit, and human.

Previous studies have demonstrated that glial cells in culture express several subtypes of functional adrenergic receptors. To determine if similar receptors are expressed by glia in vivo, we examined the expression of adrenergic receptors in the normal, crushed, and transected optic nerves of the rabbit and rat using quantitative receptor autoradiography. Additionally, we examined the expression of adrenergic receptors in the normal and damaged human optic nerve. High levels of alpha 1-, alpha 2-, beta 1-, and beta 2-adrenergic receptors were identified in the rabbit and rat forebrain. In the normal rabbit, rat, and human optic nerves, only alpha 1 and beta 2 receptors were observed, and these were present in low to moderate densities. Combined immunohistochemistry and autoradiography suggests that the majority of beta 2-adrenergic receptors in the rabbit, rat, and human optic nerve are expressed by astrocytes. After unilateral optic nerve crush or transection, only beta 2- adrenergic receptors were significantly increased. This increase in beta 2 receptors was first detectable at days 7 and 28 post-transection in the rabbit and rat, respectively. The expression of beta 2 receptors in the transected optic nerve continued to increase with time, so that by 90 d post-transection the density of beta 2 receptors in both the rabbit and rat optic nerve was among the highest of any area in the forebrain. Taken together with previous studies, these results suggest that in vivo, beta 2-adrenergic receptors may provide a therapeutic target for regulation of astrocyte functions including glycogen metabolism, cytokine release, and the hypertrophy and proliferation that occurs in response to neuronal injury.

Aged↗
PubMed1994

Substance P binding sites on intestinal lymphoid aggregates and blood vessels in inflammatory bowel disease correspond to authentic NK-1 receptors.

Previous reports have described the ectopic expression of substance P binding sites on lymphoid aggregates and small blood vessels in inflammatory bowel disease. In this report, three non-peptide NK-1 receptor antagonists, CP-96,345, RP-67,580, and L-703,606 abolished saturable 125I-Bolton-Hunter substance P binding to the ectopically expressed receptors in frozen sections of surgically resected bowel from five patients with either Crohn's disease or ulcerative colitis. The rank order of affinity was approximately substance P approximately CP-96,345 approximately L-703,606 > RP-67,580. These results suggest that: (i) the ectopically expressed substance P binding sites in inflammatory bowel disease are authentic NK-1 receptors, (ii) all ectopically expressed receptors on small blood vessels, and lymphoid aggregates as well as normally expressed receptors on the bowel circular muscle have similar receptor affinities and specificities for substance P and the non-peptide antagonists, and (iii) non-peptide antagonists may be therapeutically beneficial in inflammatory bowel disease by inhibiting the pro-inflammatory effects of substance P acting via the NK-1 receptor.

Binding Sites↗
PubMed1994

Intestinal absorption and excretion of octapeptides composed of D amino acids.

Octapeptides synthesized from D amino acids were absorbed from the intestine and excreted in urine of normal rats drinking 5% glucose/1% creatinine containing the 125I-labeled peptides at 0.1-25 mg/dl. The rats ingested fluid at the rate of about 20 ml/hr and produced urine at 15 ml/hr for several hours during the nocturnal feeding period. Sixty-one +/- 4% of the ingested creatinine and 50 +/- 3% of a lipid-insoluble D octapeptide (EASASYSA, 784 Da) were excreted intact in the urine. The steady-state molar rate of absorption-excretion of creatinine equaled or exceeded the maximum rate of carrier-mediated intestinal transport of glucose, suggesting that both the creatinine and the D octapeptide were transported paracellularly by solvent drag through absorptive cell junctions that were dilated by the glucose. More than 70% of the ingested glucose was also absorbed paracellularly. The results demonstrate that intact oligopeptides can be absorbed efficiently from the intestine when they are not hydrolyzed by membrane-bound peptidases of the brush border. The results also provide support for recent theories proposing that coupling of membrane digestion with paracellular solvent drag accounts for a major fraction of normal intestinal absorption of nutrients.

Amino Acid Sequence↗
PubMed1994

Synaptic relationship between substance P and the substance P receptor: light and electron microscopic characterization of the mismatch between neuropeptides and their receptors.

Light microscopic studies have demonstrated significant mismatches in the location of neuropeptides and their respective binding sites in the central nervous system. In the present study we used an antiserum raised against a synthetic peptide corresponding to the carboxyl-terminal tail of the substance P (SP) receptor (SPR) to further explore the relationship between a neuropeptide and its receptor. Light microscopy revealed an excellent correlation between the patterns of SPR immunoreactivity and of 125I-labeled SPR-binding sites in the central nervous system. The SPR appeared to be exclusively expressed by neurons; in fact, the SPR decorates the somatic and dendritic surface of neurons, producing Golgi-like images. Electron microscopic analysis in cortex, striatum, and spinal cord revealed that approximately 70% of the surface membrane of immunoreactive neurons is SPR laden. Simultaneous electron microscopic labeling of SP and SPR demonstrated significant mismatch at the synaptic level. Although some SP terminals contacted SPR-immunoreactive membrane, no more than 15% of the SPR-laden membrane apposed synaptic terminals. These results suggest that in contrast to more "classical" central and peripheral nervous system synapses, wherein the receptor immediately apposes the site of neurotransmitter storage and release, much of the surface of SPR-expressing neurons can be targeted by SP that diffuses a considerable distance from its site of release.

Animals↗