Diagnostic issues for conversion disorder.
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Conversion Disorder↗
Biomedical subjects
R L Martin
Publications and source records attributed to R L Martin.
Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells.
The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na+ dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-75 microM, whereas the estimated Km for putrescine uptake was only 5.36 microM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.125 microM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1,12-di(N5-azido-2-nitrobenzoyl)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.
Affinity Labels↗
Inhibition of restriction enzyme cleavage of DNA modified with 7-deaza-dGTP.
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Base Sequence↗
A simple method for calculating the dissociation constant of a receptor (or enzyme).unlabeled ligand complex from radioligand displacement measurements.
A general procedure is described for determining the dissociation constant of a receptor (or enzyme).unlabeled ligand complex (EI) by analyzing the I-dependent displacement of bound radioligand (A*) from EA*. The procedure (which involves measuring free A* in the presence of I) requires a knowledge of the total concentrations of receptor ([E]t), unlabeled ligand ([I]t) and radioligand ([A*]t), and the dissociation constant of the EA* complex. The unknown Kd is obtained from five simple, sequential calculations which are valid for either high or low affinity competitive unlabeled ligands and are independent of total receptor concentration or initial degree of saturation with A*. The procedure also provides the information needed to construct a distribution curve of all enzyme and ligand species (E, EA*, EI, A*, I) as [I]t is varied.
Enzymes↗
Adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum: ligand binding properties and the mechanism of substrate inhibition.
[35S]Adenosine-5'-phosphosulfate (APS) binding to Penicillium chrysogenum APS kinase was measured by centrifugal ultrafiltration. APS did not bind to the free enzyme with a measurable affinity even at low ionic strength where substrate inhibition by APS is quite marked. However, APS bound with an apparent Kd of 0.54 microM in the presence of 5 mM MgADP. In the presence of 0.1 M (NH4)2SO4, Kd,app was increased to 2.1 +/- 0.7 microM. Bound [35S]APS was displaced by low concentrations of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), or iso-(2') PAPS, or (less efficiently) by adenosine-3,5'-diphosphate (PAP) or adenosine-5'-monosulfate (AMS). The results support our conclusion that substrate inhibition of the fungal enzyme by APS results from the formation of a dead end E. MgADP.APS complex. That is, APS binds to the subsite vacated by PAPS in the compulsory (or predominately) ordered product release sequence (PAPS before MgADP). Radioligand displacement was used to verify the Kd for APS dissociation from E.MgADP.APS and to determine the Kd values for the dissociation of iso-PAPS (13 +/- 5 microM), PAP (4.8 mM), or AMS (5.2 mM) from their respective ternary enzyme.MgADP.ligand complexes. Incubation of the fungal enzyme with [gamma-32P]MgATP did not yield a phosphoenzyme that survives gel filtration or gel electrophoresis.
Adenosine Diphosphate↗
ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm).
ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.
Adenosine Phosphosulfate↗
The development of frequency representation in the inferior colliculus of the kitten.
While morphologically the kitten's cochlea matures first at the basal or high-frequency region, behavioural and physiological evidence suggests that it responds first to low-frequency sound. Explanations of this paradox include the suggestion that the spatial representation of frequency within the cochlea changes as a function of age. We have used the [14C]-2-deoxyglucose technique to study the development of frequency representation in the central auditory system of the kitten. We report here that while the locations within the inferior colliculus (IC) where high- and mid-frequency sounds are represented shift markedly between 10 and 35 days of age, the location where low-frequency sound is represented does not alter. The IC representation of low frequencies is adult-like by 10 days of age but that of higher frequencies continues to mature until as many as 35 days. Despite its morphological immaturity with respect to other regions, the apex of the cochlea appears to be the first region to become tuned to those frequencies to which it is tuned in the adult. We found little labelling at 5 and 7 days of age to 75-80 dB stimuli, but it is quite possible that the high-frequency region might respond to very intense low frequencies before 10 days of age. to very intense low frequencies before 10 days of age.
Acoustic Stimulation↗
Ethical issues in anesthesia: management of the do-not-resuscitate patient.
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Aged↗
Outpatient management of schizophrenia.
As effective antipsychotic pharmacotherapy has become available, patients with schizophrenia are increasingly managed in an outpatient setting by primary care physicians. Pharmacotherapy is generally effective in treating "positive," or psychotic, symptoms and lessening the risks of relapse, but ineffective in improving "negative," or deficit, symptoms. Aggressive attempts to totally control positive symptoms and to ameliorate negative symptoms tend to increase side effects and may be detrimental to the patient. Intensive psychotherapeutic and rehabilitative approaches are generally unproductive. Attempting to obtain a cure is unrealistic. A moderate approach is recommended, taking into consideration the limitations of existing treatments, achieving control of extreme symptoms and minimizing social and occupational limitations.
Administration, Oral↗
Cell cycle-dependent uptake of putrescine and its importance in regulating cell cycle phase transition in cultured adult mouse hepatocytes.
Previous studies in which investigators have induced the rate of polyamine uptake in vitro have used either inhibitors of polyamine biosynthesis or growth factors that induce cell proliferation. Recently, however, we have described the induction of putrescine uptake in cultured adult mouse hepatocytes and have shown that uptake is independent of both intracellular polyamine levels and proliferation. Although proliferation was not apparent in those studies, data suggested that, after isolation, the cells entered G1 of the cell cycle. In this study, we have examined whether the induction of putrescine uptake is a function of entry into the cell cycle and whether uptake activity is essential for optimal progression into the S phase. Using ribonuclease reductase subunit M1 as a marker of entry into the cell cycle, we have shown that hepatocytes enter G1 during the first 4 hr of culture. Both putrescine uptake and ornithine decarboxylase activity increased as the cells entered G1. Treatment of the cells with retinoic acid (10 to 33 mumol/L) prevented them from entering G1 and also inhibited the induction of the putrescine transporter by up to 90%. In contrast, initiation of G1 to S phase transition markedly down-regulated the activity of the transporter. Thus induction of the putrescine transporter in isolated hepatocytes appears to be a G1-specific event. Culturing the hepatocytes in the presence of 1,1'-bis[3-(1'-methyl-[4,4'-bipyridinium]-1-yl)-propyl]- 4,4'-bipyridinium, a potent competitive inhibitor of putrescine uptake, resulted in a 47% decrease in intracellular putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Regulation of inorganic sulfate activation in filamentous fungi. Allosteric inhibition of ATP sulfurylase by 3'-phosphoadenosine-5'-phosphosulfate.
ATP sulfurylases from Penicillium chrysogenum, Penicillium duponti, Aspergillus nidulans, and Neurospora crassa are strongly inhibited by 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of the second (adenosine-5'-phosphosulfate kinase-catalyzed) reaction in the two-step activation of inorganic sulfate. The v versus [PAPS] plots are sigmoidal. At physiological concentrations of MgATP (0.17-3 mM) and SO4(2-) (0.4-10 mM), the [I]0.5 for PAPS inhibition of the P. chrysogenum enzyme is 35-200 microM; [I]0.9 is 68-310 microM. In the presence of PAPS, the [S]0.5 values for both substrates are increased and the v versus [MgATP] and v versus [SO4(2-)] or [MoO4(2-)] plots are sigmoidal. Fluorosulfonate (FSO3-) and thiosulfate (S2O3(2-] (non-reactive sulfate analogs) inhibit the enzyme at subsaturating substrate concentrations in the absence of PAPS, but low concentrations of the analogs activate the enzyme when PAPS is present. Thus, PAPS behaves as an allosteric inhibitor of ATP sulfurylase. In contrast, adenosine-5'-phosphosulfate (APS = product Q), the immediate product of the SO4(2-)-dependent reaction, is a linear inhibitor of the P. chrysogenum enzyme, competitive with both MgATP and MoO4(2-) (Kiq = 36-73 nM). FSO3- or S2O3(2-) does not activate the enzyme in the presence of APS. The effect of PAPS on fungal ATP sulfurylase is very similar to that observed when a single highly reactive cysteinyl SH group/subunit (SH-1) is covalently modified (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288). The results suggest that in vitro SH-1 modification induces a conformational change in the enzyme that mimics the change induced in vivo by the reversible binding of PAPS. No evidence was obtained to suggest that PAPS covalently modifies SH-1. ATP sulfurylases from rat liver (Yu, M., Martin, R. L., Jain, S., Chen, L. T., and Segel, I. H. (1989) Arch. Biochem. Biophys. 269, 156-174), spinach leaf, cabbage leaf, and Saccharomyces cerevisiae are not strongly inhibited by PAPS, do not display sigmoidal initial velocity plots in the presence of PAPS, and do not contain a highly reactive cysteinyl residue whose modification induces increased [S]0.5 values and sigmoidal velocity curves. The allosteric effect of PAPS on the fungal ATP sulfurylase may be part of a sequential feedback process unique to a group of organisms that use PAPS for two diverging pathways, reductive assimilation and sulfate ester formation.
Adenine Nucleotides↗
Characterisation of putrescine uptake by cultured adult mouse hepatocytes.
Uptake of polyamines by cultured cells has been shown to be influenced by growth rate and/or differentiation. In this study, we have investigated whether the fully differentiated, non-proliferating adult mouse hepatocyte is capable of accumulating extracellular putrescine. When hepatocytes were cultured from 4 to 48 h, uptake of putrescine was found to increase substantially with time spent in culture. The Vmax for putrescine uptake increased 22-fold during this period with no change in apparent Km. Treatment of the cells with cycloheximide or actinomycin D at concentrations that did not affect cell viability inhibited the induction of putrescine uptake. Endogenous putrescine levels increased from 19.7 nmol/mg DNA after 4 h in culture to over 500 nmol/mg DNA after 48 h in culture. This increase was accompanied by a loss of over 90% of ornithine decarboxylase activity. Spermidine levels did not change over this time period, whereas spermine levels decreased by 35%. Difluoromethylornithine prevented the observed increase in intracellular putrescine but did not affect putrescine uptake. The increase in putrescine transport was not inhibited by culturing the hepatocytes in a high concentration of putrescine, spermidine or spermine. Moreover, the induction process was not stimulated by foetal calf serum but was selectively inhibited by the differentiating agents dimethylsulfoxide and retinoic acid. The results from those studies show that cultured mouse hepatocytes express a putrescine transport system that is poorly regulated by extracellular polyamines. The expression of the transporter requires the synthesis of mRNA and protein, and appears to be related to a time-dependent change in hepatocyte phenotype.
Animals↗
Kainic acid on the rat ventral medullary surface depresses hypoxic and hypercapnic ventilatory responses.
Kainic acid, topically applied to the ventral surface of the medulla immediately caudal to the trapezoid body in the urethane/chloralose anaesthetised rat, led to a depression of ventilation and a sustained rise in blood pressure; ventilatory responses to hypercapnia (10% carbon dioxide) and hypoxia (11% oxygen) were slightly depressed. Widespread application of kainic acid to an area at and slightly rostral to the rootlets of the hypoglossal nerve produced a stimulation of ventilation and an unsustained rise in blood pressure. Apnea ensued 12-28 min after application. Ventilatory responses to hypercapnia and hypoxia were markedly attenuated; more discrete bilateral application revealed two regions, one immediately rostral and lateral to the hypoglossal rootlets and the other over the point of exit of the hypoglossal nerve rootlets, which specifically contributed to the diminution of the chemosensory responses. These results raise questions about the medullary circuitry which mediates the chemoreflex regulation of breathing.
Animals↗
Alternative delivery system for controlled drugs in the surgical intensive care unit.
Faced with a serious shortage of qualified nurses for critically ill patients, methods to reduce the time required to deliver care without sacrificing quality are needed. A non-electronic device designed as a patient-controlled analgesic (PCA) was evaluated as a nurse-controlled device (NCA). Twenty-five intubated patients received morphine sulfate (MS) with the nurse-controlled device (NCA) and 12 by standard IV push policy. The average nursing time for narcotic dosing with the standard policy was 5 minutes/unit dose. A total of 1,183 NCA doses were given over 77 patient days. The average doses per patient day were 15 (2-38). The average nursing time was 22 seconds/NCA dose. The NCA saved 85 nursing minutes/patient day. Annual nursing labor costs were reduced by $77,000.00 with NCA. Total costs for standard IV push narcotic use were $36.43/patient day versus $35.45/patient day for NCA. Using this protocol, the NCA system saved $8,500.00 annually. By increasing the duration of PCA use to 72 hours, the annual savings would become $49,500.00. These data indicate that a simple NCA can deliver controlled drugs rapidly and safely, save valuable nursing time, and decrease the cost of ICU care.
Economics, Nursing↗
Antibody production and growth of mouse hybridoma cells in Nutridoma media supplements.
Traditionally, cell culturists have relied upon the addition of serum to culture medium for the growth and maintenance of cell lines. However, many aspects of the use of serum in tissue culture are problematic. Cell culture supplements that circumvent the need for serum are readily available and provide a consistent protein composition. This defined environment allows the antibody to be more easily purified from culture supernatants. Nutridoma media supplements were formulated to support the growth of lymphoblastoid cells in a defined culture environment. In this study, Nutridoma media supplements were tested in parallel with serum-containing cultures to determine if Nutridoma supplemented medium is effective in supporting hybridoma cell growth and antibody production in three hybridoma cell lines. Data, based on cell growth and antibody production, show the importance of basal media selection when serum is replaced with Nutridoma media supplements. SDS-PAGE results show that cell supernatants from Nutridoma supplemented cultures contain very few contaminating proteins.
Animals↗
Inhibition of putrescine uptake by polypyridinium quaternary salts in B16 melanoma cells treated with difluoromethylornithine.
Several bipyridinium, tetrapyridinium and hexapyridinium quaternary salts have been found to be potent inhibitors of putrescine uptake into B16 melanoma cells which had previously been treated with difluoromethylornithine. In general, the potency of inhibitors increased as the number of quaternary centres increased. A relationship between the distance apart of the positively charged nitrogen atoms and the potency of the salts as inhibitors of uptake has been established by comparison with a number of diaminoalkanes. It was found that an inter-nitrogen distance of 0.6-0.7 nm or 1.0-1.1 nm was optimal for high activity. This finding is significant in determining structural features of the polyamine transport system.
Animals↗
The "regulatory" sulfhydryl group of Penicillium chrysogenum ATP sulfurylase. Cooperative ligand binding after SH modification; chemical and thermodynamic properties.
ATP sulfurylase from Penicillium chrysogenum is a homohexamer that contains three free sulfhydryl groups/subunit, only one of which (designated SH-1) can be modified by disulfide, maleimide, and halide reagents under nondenaturing conditions. Modification of SH-1 has only a small effect on kcat but causes the [S]0.5 values for MgATP and SO4(2-) (or MoO4(2-) to increase by an order of magnitude. Additionally, the velocity curves become sigmoidal with a Hill coefficient (nH) of about 2 (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288). Direct equilibrium binding measurements confirmed that [32P]MgATP binds to the SH-modified enzyme in a positively cooperative fashion (nH = 2.0) if a sulfate subsite ligand (e.g. FSO3-) is also present. [35S]Adenosine 5'-phosphosulfate (APS) binding to the SH-modified enzyme displayed positive cooperativity (nH = 1.9) in the absence of a PPi subsite ligand. The results indicate that positive cooperativity requires occupancy of the adenylyl and sulfate (but not the pyrophosphate) subsites. [35S]APS binding to the native enzyme displayed negative cooperativity (or binding to at least two classes of sites). Isotope trapping profiles for the single turnover of [35S]APS: (a) confirmed the equilibrium binding curves, (b) indicated that all six sites/hexamer are catalytically active, and (c) showed that APS does not dissociate at a significant rate from E.APS.PPi. The MgPPi concentration dependence of [35S]APS trapping was indicative of MgPPi binding to two classes of sites on both the native and SH-modified enzyme. Inactivation of the native or SH-modified enzyme by phenylglyoxal in the presence of saturating APS was biphasic. The semilog plots suggested that only half of the sites were highly protected. The cumulative data suggest a model in which pairs of sites or subunits can exist in three different states designated HH (both sites have a high APS affinity, as in the native free enzyme), LL (both sites have a low APS affinity as in the SH-modified enzyme), and LH (as in the APS-occupied native or SH-modified enzyme). Thus, the HH----LH transition displays negative cooperativity for APS binding while the LL----LH transition displays positive cooperativity. The relative reactivities of like-paired SH-reactive reagents were in the order: N-phenylmaleimide greater than N-ethylmaleimide; dithionitropyridine greater than dithionitrobenzoate; thiolyte-MQ greater than thiolyte-MB. The log kmod versus pH curve indicates that the pKa of SH-1 is greater than 9.(ABSTRACT TRUNCATED AT 400 WORDS)
Hydrogen-Ion Concentration↗
Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase.adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase.
At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no "substrate channeling" of APS between the two sulfate-activating enzymes. APS kinase had no effect on the [S]0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound [35S]APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a "3'-phosphoadenosine 5'-phosphosulfate synthetase" complex.
Adenosine Monophosphate↗