Biomedical subjects

A I Spielman

Publications and source records attributed to A I Spielman.

At least 19 recordsLinked to original sources

PubMed1999

Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles.

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca2+ with little or no effect on Vmax(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca2+ is larger, while the increase in Vmax(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca2+ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca2+ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 micrometer ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in Vmax(Ca) in addition to an increase in the pump's apparent affinity for Ca2+ in response to phosphorylation of PLN by protein kinase A.

Adenosine Triphosphate↗

PubMed1999

Ggamma13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium.

Gustducin is a transducin-like G protein selectively expressed in taste receptor cells. The alpha subunit of gustducin (alpha-gustducin) is critical for transduction of responses to bitter or sweet compounds. We identified a G-protein gamma subunit (Ggamma13) that colocalized with alpha-gustducin in taste receptor cells. Of 19 alpha-gustducin/Ggamma13-positive taste receptor cells profiled, all expressed the G protein beta3 subunit (Gbeta3); approximately 80% also expressed Gbeta1. Gustducin heterotrimers (alpha-gustducin/Gbeta1/Ggamma13) were activated by taste cell membranes plus bitter denatonium. Antibodies against Ggamma13 blocked the denatonium-induced increase of inositol trisphosphate (IP3) in taste tissue. We conclude that gustducin heterotrimers transduce responses to bitter and sweet compounds via alpha-gustducin's regulation of phosphodiesterase (PDE) and Gbetagamma's activation of phospholipase C (PLC).

Amino Acid Sequence↗

PubMed1999

Ceramide triggers intracellular calcium release via the IP(3) receptor in Xenopus laevis oocytes.

Ceramide, a product of sphingomyelin turnover, is a lipid second messenger that mediates diverse signaling pathways, including those leading to cell cycle arrest and differentiation. The mechanism(s) by which ceramide signals downstream events have not been fully elucidated. Here we show that, in Xenopus laevis oocytes, ceramide-induced maturation is associated with the release of intracellular calcium stores. Ceramide caused a dose-dependent elevation in the second messenger inositol 1,4,5-trisphosphate (IP(3)) via activation of G(q/11)alpha and phospholipase C-betaX. Elevation of IP(3), in turn, activated the IP(3) receptor calcium release channel on the endoplasmic reticulum, resulting in a rise in cytoplasmic calcium. Thus our study demonstrates that cross talk between the ceramide and phosphoinositide signaling pathways modulates intracellular calcium homeostasis.

Animals↗

PubMed1999

Possible novel mechanism for bitter taste mediated through cGMP.

Taste is the least understood among sensory systems, and bitter taste mechanisms pose a special challenge because they are elicited by a large variety of compounds. We studied bitter taste signal transduction with the quench-flow method and monitored the rapid kinetics of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) production and degradation in mouse taste tissue. In response to the bitter stimulants, caffeine and theophylline but not strychnine or denatonium cGMP levels demonstrated a rapid and transient increase that peaked at 50 ms and gradually declined throughout the following 4.5 s. The theophylline- and caffeine-induced effect was rapid, transient, concentration dependent and gustatory tissue-specific. The effect could be partially suppressed in the presence of the soluble guanylyl cyclase (GC) inhibitor 10 microM ODQ and 30 microM methylene blue but not 50 microM LY 83583 and boosted by nitric oxide donors 25 microM NOR-3 or 100 microM sodium nitroprusside. The proposed mechanism for this novel cGMP-mediated bitter taste signal transduction is cGMP production partially by the soluble GC and caffeine-induced inhibition of one or several phosphodiesterases.

Aminoquinolines↗

PubMed1999

Interactions of 6-gingerol and ellagic acid with the cardiac sarcoplasmic reticulum Ca2+-ATPase.

The inotropic/lusitropic effects of beta-adrenergic agonists on the heart are mediated largely by protein kinase A (PKA)-catalyzed phosphorylation of phospholamban, the natural protein regulator of the Ca2+ pump present in sarcoplasmic reticulum (SR) membranes. Gingerol, a plant derivative, is known to produce similar effects when tested in isolated cardiac muscle. The purpose of the present study was to compare the effects of gingerol and another plant derivative, ellagic acid, on the kinetics of the SR Ca2+ pump with those of PKA-catalyzed phospholamban phosphorylation to elucidate their mechanisms of Ca2+ pump regulation. As previously demonstrated for PKA, 50 microM gingerol or ellagic acid increased Vmax(Ca) of Ca2+ uptake and Ca2+-ATPase activity assayed at millimolar ATP concentrations in light cardiac SR vesicles. Unlike PKA, which decreases Km(Ca), neither compound had a significant effect on Km(Ca) in unphosphorylated vesicles. However, gingerol increased Km(Ca) in phosphorylated vesicles, in which Ca2+ uptake was significantly increased further at saturating Ca2+ and remained unchanged at subsaturating Ca2+. An inhibition of Ca2+ uptake by gingerol at micromolar MgATP concentrations was overcome with increasing MgATP concentrations. The stimulation of Ca2+ uptake attributable to gingerol in unphosphorylated microsomes at saturating Ca2+ was 30% to 40% when assayed at 0.05 to 2 mM MgATP and only about 12% in phosphorylated microsomes as well as in rabbit fast skeletal muscle light SR. The present results support the view that an ATP-dependent increase in Vmax(Ca) of the SR Ca2+ pump plays an important role in mediating cardiac contractile responses to gingerol and phospholamban-dependent beta-adrenergic stimulation.

Animals↗

PubMed1998

The arginine taste receptor. Physiology, biochemistry, and immunohistochemistry.

The amino acid, L-arginine (L-Arg), is a potent taste stimulus for the channel catfish, Ictalurus punctatus. Receptor binding studies demonstrated a high-affinity binding of L-Arg to putative taste receptor sites. This binding could be inhibited by preincubation of the tissue in the lectins Phaseolus vulgaris agglutinin (PHA) and Ricinus communis agglutinin I (RCA I). Neurophysiological studies demonstrated that the L-Arg receptor is a stimulus-gated ion channel type receptor whose conductance was stimulated by L-Arg and inhibited by D-arginine (D-Arg). To purify the receptor we subjected CHAPS solubilized partial membrane preparation from barbel epithelium to RCA I lectin affinity chromatography. The bound proteins were eluted with D-galactose. When these proteins were reconstituted into lipid bilayers, L-Arg activated single channel currents with conductances between 45 and 85 pS. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted protein showed a distinct band at approximately 83 kDa. Polyclonal antibodies raised against this 83-kDa band in guinea pigs reacted with numerous small (approximately 1 micron) sites within the taste pore of every taste bud when applied to fixed nonpermeabilized barbels. This observation suggests that the antibodies recognize an externally-facing epitope of the putative Arg receptor. The antibodies also inhibited L-Arg-stimulated currents in reconstitution studies. Sephacryl S-300 HR chromatography of the eluant from the affinity column showed a high molecular weight peak (> 700 kDa) which was recognized by the antibodies. Reconstitution of the protein from this peak into a lipid bilayer resulted in L-Arg-stimulated channels that could be inhibited by D-Arg. This high molecular weight component may be aggregates of the arginine taste receptor.

Animals↗

PubMed1998

Identification and immunohistochemical localization of protein precursors to human axillary odors in apocrine glands and secretions.

OBJECTIVES: To determine the cellular localization in male and female axillary tissue for apocrine secretion odor-binding proteins 1 (ASOB1) and 2 (ASOB2) and the electrophoretic pattern of female apocrine proteins and to begin characterization of the ASOB1 protein. DESIGN: Immunohistochemical techniques were used with biopsy samples from axillary tissue of male and female subjects. Immunological techniques and microsequencing were used to characterize several of the proteins in male and female apocrine secretions. SETTING: A university medical center. PARTICIPANTS: Healthy male and female volunteers who donated apocrine secretions and/or axillary tissue. RESULTS: Specific immunoreactivity was localized only to the apocrine glands in both sexes. Furthermore, only preabsorption with a mixed apocrine secretion sample eliminated all immunoreactivity. The electrophoretic pattern of proteins in female apocrine secretions is similar to that in male secretions. Western blotting of the separated proteins from female samples using serum samples containing antibodies to ASOB1 and ASOB2 yielded identical results to those found with separated proteins from male samples. Partial sequence data obtained from the N-terminus of ASOB1 suggested that it shares homology with the alpha-chain of apolipoprotein J (Apo J). Apocrine secretion odor-binding protein 1 is not immunologically similar to ApoJ, but 2 other apocrine secretion proteins are. CONCLUSIONS: Male and female subjects appear to have the same glycoprotein carriers for (E)-3-methyl-2-hexenoic acid localized to the apocrine glands. The N-terminal sequence for ASOB1 may be homologous to Apo J, but it is not immunologically similar to it. However, 2 other proteins in the apocrine secretion appear to be the monomer and dimer forms of Apo J.

Adult↗

PubMed1998

Gustducin and its role in taste.

The mechanisms responsible for taste signal transductions are very complex. A key molecule, alpha-gustducin, a primarily taste-specific G protein alpha-subunit, was discovered in 1992 and was later found to be involved in both bitter and sweet taste transduction. A proposed mechanism for alpha-gustducin involves coupling specific cell-surface receptors with a cyclic nucleotide phosphodiesterase which would open a cyclic nucleotide-suppressible cation channel leading to influx of calcium, and ultimately leading to release of neurotransmitter. Although "knock-out" animals deficient in the alpha-gustducin gene clearly demonstrate that gustducin is an essential molecule for tasting certain bitter and sweet compounds, the precise role of alpha-gustducin in bitter and sweet taste is presently unclear. Indeed, there are several other signaling mechanisms in sweet and bitter taste, apparently unrelated to alpha-gustducin, that increase cyclic AMP or inositol 1,4,5 trisphosphate. Thus, proposed models for alpha-gustducin and those found by other laboratories may be parallel and interdependent.

Animals↗

PubMed1998

What's in a name?

Explore the source record for details and available documents.

Dental Research↗

PubMed1998

Chemosensory function and dysfunction.

Taste and smell are fundamental sensory systems essential in nutrition and food selection, for the hedonic and sensory experience of food, for efficient metabolism, and, in general, for the maintenance of a good quality of life. The gustatory and olfactory systems demonstrate a diversity of transduction mechanisms, and during the last decade, considerable progress has been made toward our understanding of the basic mechanisms of taste and smell. Understanding normal chemosensory function helps clarify the molecular events that underlie taste and smell disorders. At least 2,000,000 Americans suffer from chemosensory disorders--a number that is likely to grow as the aging segment of the population increases. Smell disorders are more frequent than taste disturbances, due to the vulnerability and anatomical distinctiveness of the olfactory system, and because a decline in olfactory function is part of the normal aging process. Common gustatory and olfactory complaints are due to a number of medications, to upper respiratory infections, to nasal and paranasal sinus diseases, and to damage to peripheral nerves supplying taste and smell. Most chemosensory complaints have an identifiable cause. Although diagnosis of taste and smell disorders has improved considerably over the last two decades, treatment of these disorders is still limited to conditions with discernible and reversible causes. Future research is needed for a better understanding of chemosensory mechanisms, establishing improved diagnostic procedures, and disseminating knowledge on chemosensory disorders among practitioners and the general public.

Aging↗

PubMed1997

Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes.

Protein kinase A- (PKA-) catalyzed phosphorylation of phospholamban (PLN), the protein regulator of the cardiac Ca pump, mediates abbreviation of systole in response to beta-adrenergic agonists. Investigators previously, however, have been unsuccessful in demonstrating an effect of PLN phosphorylation or anti-PLN monoclonal antibody (mAb), which is considered to mimic phosphorylation's well-known effect on Km(Ca), on microsomal Ca uptake at the (high) Ca2+ concentrations found intracellularly at peak systole. We therefore compared the effects of the catalytic subunit of PKA and anti-PLN mAb on the kinetics of Ca uptake in sucrose gradient-purified cardiac microsomes. Both treatments produced a 33-44% increase in Vmax(Ca) at 25 and 37 degrees C, and an 11-31% decrease in Km(Ca) with comparable changes in Ca2+-ATPase activity. An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increased Vmax(Ca) of Ca uptake at 25 degrees C but not at 37 degrees C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from Pi at different temperatures. Our data document almost identical increases in Vmax(Ca) of microsomal Ca uptake with PLN phosphorylation or addition of anti-PLN mAb and hence provide insight into the kinetic mechanism of PLN's regulation of the cardiac sarcoplasmic reticulum Ca pump protein.

Animals↗

PubMed1997

Comparison of the effects of phospholamban and jasmone on the calcium pump of cardiac sarcoplasmic reticulum. Evidence for modulation by phospholamban of both Ca2+ affinity and Vmax (Ca) of calcium transport.

Regulation of the calcium pump of the cardiac sarcoplasmic reticulum by phosphorylation/dephosphorylation of phospholamban is central to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. In order to study the mechanism of this regulation, we first obtained purified ruthenium red-insensitive microsomes enriched in sarcoplasmic reticulum membranes. The kinetics of microsomal Ca2+ uptake after phospholamban phosphorylation or trypsin treatment, which cleaves the inhibitory cytoplasmic domain of phospholamban, were then compared with those in the presence of jasmone, whose effects on the kinetics of fast skeletal muscle Ca2+-ATPase are largely known. All three treatments increased Vmax (Ca) at 25 degrees C and millimolar ATP; phosphorylation and trypsin decreased the Km (Ca), while jasmone increased it. Trypsin and jasmone increased the rate of E2P decomposition 1.8- and 3. 0-fold, respectively. The effects of phospholamban phosphorylation and jasmone on the Ca2+-ATPase activity paralleled their effects on Ca2+ uptake. Our data demonstrate that phospholamban regulates E2P decomposition in addition to the known increase in the rate of a conformational change in the Ca2+-ATPase upon binding the first of two Ca2+. These steps in the catalytic cycle of the Ca2+-ATPase may contribute to or account for phospholamban's effects on both Vmax (Ca) and Km (Ca), whose relative magnitude may vary under different experimental and, presumably, physiological conditions.

Adenosine Triphosphate↗

PubMed1996

A human axillary odorant is carried by apolipoprotein D.

The characterization of the source of the odor in the human axillary region is not only of commercial interest but is also important biologically because axillary extracts can alter the length and timing of the female menstrual cycle. In males, the most abundant odor component is known to be E-3-methyl-2-hexenoic acid (E-3M2H), which is liberated from nonodorous apocrine secretions by axillary microorganisms. Recently, it was found that in the apocrine gland secretions, 3M2H is carried to the skin surface bound to two proteins, apocrine secretion odor-binding proteins 1 and 2 (ASOB1 and ASOB2) with apparent molecular masses of 45 kDa and 26 kDa, respectively. To better understand the formation of axillary odors and the structural relationship between 3M2H and its carrier protein, the amino acid sequence and glycosylation pattern of ASOB2 were determined by mass spectrometry. The ASOB2 protein was identified as apolipoprotein D (apoD), a known member of the alpha2mu-microglobulin superfamily of carrier proteins also known as lipocalins. The pattern of glycosylation for axillary apoD differs from that reported for plasma apoD, suggesting different sites of expression for the two glycoproteins. In situ hybridization of an oligonucleotide probe against apoD mRNA with axillary tissue demonstrates that the message for synthesis of this protein is specific to the apocrine glands. These results suggest a remarkable similarity between human axillary secretions and nonhuman mammalian odor sources, where lipocalins have been shown to carry the odoriferous signals used in pheromonal communication.

Adult↗

PubMed1996

Rapid kinetics of second messenger production in bitter taste.

The tasting of bitter compounds may have evolved as a protective mechanism against ingestion of potentially harmful substances. We have identified second messengers involved in bitter taste and show here for the first time that they are rapid and transient. Using a quench-flow system, we have studied bitter taste signal transduction in a pair of mouse strains that differ in their ability to taste the bitter stimulus sucrose octaacetate (SOA); however, both strains taste the bitter agent denatonium. In both strains of mice, denatonium (10 mM) induced a transient and rapid increase in levels of the second messenger inositol 1,4,5-trisphosphate (IP3) with a maximal production near 75-100 ms after stimulation. In contrast, SOA (100 microM) brought about a similar increase in IP3 only in SOA-taster mice. The response to SOA was potentiated in the presence of GTP (1 microM). The GTP-enhanced SOA-response supports a G protein-mediated response for this bitter compound. The rapid kinetics, transient nature, and specificity of the bitter taste stimulus-induced IP3 formation are consistent with the role of IP3 as a second messenger in the chemoelectrical transduction of bitter taste.

Animals↗

PubMed1996

Regulation of extracellular calcium sensing in rat osteoclasts by femtomolar calcitonin concentrations.

Certain eukaryotic cells can sense changes in their extracellular Ca2+ concentration through molecular structures termed Ca(2+)-sensing receptors (CaRs). We have shown recently that in the bone-resorbing osteoclast, a unique cell surface-expressed ryanodine receptor (RyR), functions as the CaR. The present study demonstrates that the sensitivity of this receptor is modulated by physiological femtomolar concentrations of the bone-conserving hormone, calcitonin. Calcitonin was found to inhibit cytosolic Ca2+ responses to both Ca2+ and Ni2+. The latter inhibition was mimicked by amylin (10(-12) M), calcitonin gene-related peptide (10(-12) M), cholera toxin (5 micrograms/l) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) (2.5 x 10(-4) or 5 x 10(-4) M) and was reversed by the protein kinase A phosphorylation inhibitor, IP-20. Finally, using a quench flow module, we showed that cellular cAMP levels rise to a peak within 25 ms of calcitonin application; this is consistent with the peptide's rapid effect on CaR activation. We conclude, therefore, that cAMP plays a critical role in the control of CaR function by calcitonin.

Animals↗

PubMed1996

Halitosis. A common oral problem.

Halitosis is caused primarily by bacterial putrefaction and the generation of volatile sulfur compounds. Ninety percent of patients suffering from halitosis have oral causes, such as poor oral hygiene, periodontal disease, tongue coat, food impaction, unclean dentures, faulty restorations, oral carcinomas, and throat infections. The remaining 10 percent of halitosis sufferers have systemic causes that include renal or hepatic failure, carcinomas, diabetes or trimethylaminuria. Modern analytical and microbiological techniques permit diagnosis of bad breath. Management of halitosis involves maintaining proper oral hygiene, and periodontal treatment, including tongue brushing.

Bacteria↗

PubMed1995

Proteinaceous precursors of human axillary odor: isolation of two novel odor-binding proteins.

The characteristic odor which arises in the human axillary region consists of volatile C6-C11 acids with the most abundant being (E)-3-methyl-2-hexenoic acid (E-3M2H). This acid, as well as several other components of the characteristic axillary odor, can be liberated from the odorless, aqueous soluble components of apocrine secretion by either saponification or bacteriolysis. It is therefore likely that a major characteristic odor is being carried to the skin surface bound to a water soluble precursor where it is liberated by axillary bacteria. The individual proteins found in apocrine secretions were separated, isolated and hydrolyzed with the resultant hydrolyzates analyzed by gas chromatography/mass spectrometry. These studies demonstrated that 3M2H was liberated from 2 proteins with apparent molecular mass of 26 and 45 kilodaltons: Apocrine Secretion Odor-Binding Protein 1 and 2, respectively (ASOB1 and ASOB2). Antisera to these proteins were prepared and used to examine a variety of other body fluids. Several fluids contained an immunoreactive protein with the same electropheretic migration pattern as the 45 KDa protein. Three of these body fluids (tears, nasal secretions and saliva) were separated into aqueous and organic soluble fractions and hydrolyzed to demonstrate that 3M2H could be liberated from the aqueous soluble materials. These results suggest interesting parallels between non-human mammalian odors used as chemical signals and human axillary odor. Previous studies have suggested the axillae as a source of human primer-type pheromones; consequently, if the odors which characterize the underarm are responsible for the pheromonal activity, then the chemistry involved may be similar to that in other mammalian chemical communication systems where proteins act as carriers of one or more chemical signals.

Adult↗

PubMed1994

Generation of inositol phosphates in bitter taste transduction.

It is probable that there is a diversity of mechanisms involved in the transduction of bitter taste. One of these mechanisms uses the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Partial membrane preparations from circumvallate and foliate taste regions of mice tongues responded to the addition of known bitter taste stimuli by increasing the amount of inositol phosphates produced after 30 s incubation. Addition of both the bitter stimulus, sucrose octaacetate and the G-protein stimulant, GTP gamma S, led to an enhanced production of inositol phosphates compared with either alone. Pretreatment of the tissue samples with pertussis toxin eliminated all response to sucrose octaacetate plus GTP gamma S, whereas pretreatment with cholera toxin was without effect. Western blots of solubilized tissue from circumvallate and foliate regions probed with antibodies to the alpha-subunit of several types of G-proteins revealed bands reactive to antibodies against G alpha i1-2 and G alpha o, with no apparent activity to antibodies against G alpha i3. Given the results from the immunoblots and those of the toxin experiments, it is proposed that the transduction of the bitter taste of sucrose octaacetate in mice involves a receptor-mediated activation of a Gi-type protein which activates a phospholipase C to produce the two second messengers, IP3 and DAG.

Animals↗