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Biomedical subjects

J R Stone

Publications and source records attributed to J R Stone.

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

Spectral and ligand-binding properties of an unusual hemoprotein, the ferric form of soluble guanylate cyclase.

The soluble form of guanylate cyclase (sGC) is a hemoprotein which serves as the only known receptor for the signaling agent nitric oxide (.NO). The enzyme is a heterodimer in which each subunit binds 1 equiv of 5-coordinate high-spin type b heme. .NO increases the Vmax of sGC up to 400-fold by binding to the heme to form a 5-coordinate ferrous nitrosyl complex. The electron paramagnetic resonance spectrum of the ferric form of the enzyme has been obtained. The spectrum displays rhombic symmetry and is indicative of a high-spin heme. Computer simulation of the EPR spectrum yields g values of 6.36, 5.16, and 2.0 with linewidths of 3.3, 4.1, and 3.3 mT, respectively. Using electronic absorption spectroscopy, it was observed that the ferric heme binds cyanide to form a 6-coordinate low-spin complex. The rate constants for association (k(on)) and dissociation (k(off)) of cyanide at 10 degrees C have been determined to be (7.8 +/- 0.3) x 10(-2) M(-1) s(- 1) and (7.2 +/- 0.2) x 10(-5) s(-1), respectively. Unlike the ferrous form of the enzyme, which has a low affinity for ligands that form 6-coordinate complexes due to an unusually fast off-rate, the ferric form of the enzyme appears to have a low affinity for ligands due to a slow on-rate. The ferric heme binds azide with a Kd of 26 +/- 4 mM to form a high-spin complex. The ferric form of the enzyme has a specific activity of approximately 57% that of the nonactivated ferrous form of the enzyme. However, in contrast to the mild activation of the ferrous enzyme by carbon monoxide, the ferric enzyme is not activated by cyanide. These results indicate that there may be a significant structural change in the protein upon the oxidation of the heme iron.

Azides↗
PubMed1996

Binding of nitric oxide and carbon monoxide to soluble guanylate cyclase as observed with Resonance raman spectroscopy.

Resonance Raman spectra have been recorded for the ferrous heme of soluble guanylate cyclase (sGC), the only receptor known thus far for .NO. On the basis of the frequencies of porphyrin core sensitive vibrations in the high frequency region of the Raman spectrum, we conclude that the ferrous heme is five-coordinate, high spin, when no exogenous ligands are present. We assign a prominent vibration that occurs at 204 cm-1 in the reduced enzyme to the heme Fe(2+)-proximal histidine stretching vibration. In the .NO bound form of the enzyme, the heme Fe2+ retains a five-coordinate geometry. Assuming that .NO binds to the distal side of the heme, this observation indicates that the weak Fe-His bond breaks when .NO binds. Two isotope-sensitive vibrations are observed in the .NO bound enzyme, one at 1677 cm-1, attributed to the N-O stretching vibration, and one at 525 cm-1, attributed to the Fe-NO stretching vibration. When CO is bound to the ferrous heme, the heme ligation is six-coordinate. From this, we conclude that the Fe-His bond is still intact and that, if cleavage of the Fe-proximal ligand bond is necessary for complete activation of sGC, then CO should only weakly activate the enzyme, which has been shown to be the case. In the carbonmonoxy enzyme, the Fe-CO stretching vibration is observed at 472 cm-1 and the Fe-C-O bending vibration is detected at 562 cm-1. These frequencies are the lowest yet observed for the Fe-CO stretching and Fe-C-O bending modes in heme proteins or model systems with imidazole as the proximal ligand and suggest that there is significant negative polarity in the distal pocket. The negative polarity and the low frequency of the Fe-His stretching vibration may account for the very low O2 affinity of sGC.

Animals↗
PubMed1996

Spectral and kinetic studies on the activation of soluble guanylate cyclase by nitric oxide.

The soluble form of guanylate cyclase (sGC) is the only definitive receptor for the signaling agent nitric oxide (.NO). The enzyme is a heterodimer of homologous subunits in which each subunit binds 1 equiv of 5-coordinate high-spin heme. .NO increases the Vmax of sGC up to 400-fold and has previously been shown to bind to the heme to form a 5-coordinate complex. Using stopped-flow spectrophotometry, it is demonstrated that the binding of .NO to the heme of sGC is a complex process. .NO first binds to the heme to form a 6-coordinate nitrosyl complex, which then converts to a 5-coordinate nitrosyl complex through one of two ways. For 28 +/- 4% of the heme, the 6-coordinate nitrosyl complex rapidly (approximately 20 s-1) converts to the 5-coordinate complex. For the remaining 72 +/- 4% of the heme, the conversion of the 6-coordinate nitrosyl complex to a 5-coordinate nitrosyl complex is slow (0.1-1.0 s-1) and is dependent upon the interaction of .NO with an unidentified non-heme site on the protein. The heme (200 nM) was completely converted to the 5-coordinate state with as little as 500 nM .NO, and the equilibrium dissociation constant of .NO for activating the enzyme was determined to be < or = 250 nM. Gel-filtration analysis indicates that the binding of .NO to the heme has no effect on the native molecular mass of the protein. Correlation of electronic absorption spectra with activity measurements indicates that the 5-coordinate nitrosyl form of the enzyme is activated relative to the resting 5-coordinate ferrous form of the enzyme.

Animals↗
PubMed1995

The ferrous heme of soluble guanylate cyclase: formation of hexacoordinate complexes with carbon monoxide and nitrosomethane.

The soluble form of guanylate cyclase (sGC) is the only definitive receptor for the signaling agent nitric oxide (.NO). The enzyme is a homologous heterodimer in which each subunit binds 1 equiv of 5-coordinate high-spin heme. .NO increases the Vmax of sGC up to 400-fold, probably by binding directly to the heme. Carbon monoxide (CO) forms a 6-coordinate complex with the heme and weakly activates the enzyme. Using stopped-flow spectrophotometry, the on-rate and off-rate for the binding of CO to the heme have been determined to be (3.58 +/- 0.15) x 10(4) M-1 s-1 and 3.5 +/- 0.5 s-1, respectively, at 10 degrees C. The equilibrium dissociation constant (Kd) has been independently determined to be 97 +/- 9 microM. Comparison of this Kd with that calculated from the rate constants indicates that the binding of CO to sGC is a simple one-step process, in which the off-rate of CO from the hexacoordinate complex is much faster than typically found in hemoproteins. The Kd of CO for activating the enzyme was also determined and compared to that for binding to the heme. Nitrosomethane forms irreversible complexes with typical ferrous hemoproteins but was observed to bind reversibly to the heme in sGC, with an off-rate > or = (7.6 +/- 0.2) x 10(-3) s-1. In general, the ferrous heme in sGC has a low affinity for ligands that form 6-coordinate complexes due primarily to fast ligand off-rates.

Animals↗
PubMed1995

Heme stoichiometry of heterodimeric soluble guanylate cyclase.

The soluble form of guanylate cyclase (sGC) is to date the only definitive receptor for the novel signaling agent nitric oxide (.NO). .NO increases the Vmax of sGC by 100-200-fold, and it has been proposed that this activation occurs subsequent to the binding of .NO to a heme moiety on the enzyme. It has previously been demonstrated that the enzyme can be purified in a state containing as much as 1 heme per heterodimer. However, since the two subunits of the heterodimer display considerable homology, and the enzyme routinely loses heme upon purification, it has been unclear whether the native heme stoichiometry is 1 per heterodimer or 2 per heterodimer. Using a novel procedure, the enzyme has been purified to homogeneity from bovine lung in a state containing 1.52 +/- 0.10 equiv of heme per heterodimer, indicating that the native heme stoichiometry is 2 per heterodimer. The .NO-activated specific activity of this enzyme is increased by 50% over that of enzyme containing 1 heme per heterodimer and is the highest specific activity ever observed for sGC. Spectrally only one type of heme is observed, indicating that both hemes in the heterodimer are in similar environments. It is concluded that each subunit of the heterodimer binds 1 equiv of heme at a site conserved between the two subunits. Alignment of the nine published cDNA sequences for sGC indicates that the heme binding domain is the central portion of each subunit, corresponding to residues 213-370 in the bovine beta 1 sequence.

Amino Acid Sequence↗
PubMed1995

Electron paramagnetic resonance spectral evidence for the formation of a pentacoordinate nitrosyl-heme complex on soluble guanylate cyclase.

Nitric oxide (.NO) is an important intercellular signaling agent throughout the animal kingdom. The majority of the effects of .NO is due to the direct stimulation of soluble guanylate cyclase (sGC). It has been proposed that .NO activates sGC by interacting with a heme moiety on the enzyme. The electron paramagnetic resonance (EPR) spectra of the ferrous-[14N/15N]nitrosyl complexes of the purified enzyme have been obtained. The spectrum of the [14N]nitrosyl-sGC complex was almost identical to that of a 5-coordinate. Hyperfine coupling constants and g-values were determined from computer simulations of the EPR spectra. Electronic absorption spectroscopy was used to show that the 5-coordinate nitrosyl complex on sGC forms under conditions that are typically used to assay activation of the enzyme.

Computer Simulation↗
PubMed1995

Practice parameters for intravenous analgesia and sedation for adult patients in the intensive care unit: an executive summary. Society of Critical Care Medicine.

OBJECTIVE: The development of practice parameters for intravenous analgesia and sedation for adult patients in the intensive care unit (ICU) setting for the purpose of guiding clinical practice. PARTICIPANTS: A task force of more than 40 experts in disciplines related to the use of analgesic and sedative agents in the ICU was convened from the membership of the American College of Critical Care Medicine (ACCM) and the Society of Critical Care Medicine (SCCM). EVIDENCE: The task force members provided the personal experience and determined the published literature (MEDLINE articles, textbooks, pharmacopeias, etc.) from which consensus would be sought. Published literature was reviewed and classified into one of four predetermined categories, according to study design and scientific value. CONSENSUS PROCESS: The task force met several times as a whole, and numerous times in smaller groups by teleconference, over a 1-yr period to identify the pertinent literature and arrive at consensus recommendations for the whole task force to discuss. Consideration was given to the relationship between the weight of scientific information and the experts' viewpoints. Over the next year, draft documents were composed by a task force steering committee and debated by the task force members until consensus was reached by nominal group process. The task force draft was then reviewed, assessed, and edited by the Board of Regents of the ACCM. After steering committee approval, the draft document was reviewed and approved by the SCCM Council. DATA SYNTHESIS: To facilitate rapid communication of the six recommendations contained within the complete and unabridged practice parameter document, an executive summary was prepared for publication by the ACCM Board of Regents, and this executive summary was approved by the task force steering committee and the SCCM Executive Council. CONCLUSIONS: A consensus of experts provided six recommendations with supporting data for intravenous analgesia and sedation in the ICU setting: a) morphine sulfate is the preferred analgesic agent for critically ill patients; b) fentanyl is the preferred analgesic agent for critically ill patients with hemodynamic instability, for patients manifesting symptoms of histamine release with morphine, or morphine allergy; c) hydromorphone can serve as an acceptable alternative to morphine; d) midazolam or propofol are the preferred agents only for the short-term (< 24 hrs) treatment of anxiety in the critically ill adult; e) lorazepam is the preferred agent for the prolonged treatment of anxiety in the critically ill adult; f) haloperidol is the preferred agent for the treatment of delirium in the critically ill adult. This executive summary selectively presents supporting information and is not intended as a substitute for the complete document.

Adult↗
PubMed1995

Practice parameters for sustained neuromuscular blockade in the adult critically ill patient: an executive summary. Society of Critical Care Medicine.

OBJECTIVE: The development of practice parameters for achieving sustained neuromuscular blockade in the adult critically ill patient for the purpose of guiding clinical practice. PARTICIPANTS: A task force of more than 40 experts in disciplines related to the use of neuromuscular blocking agents in the intensive care unit was convened from the membership of the American College of Critical Care Medicine (ACCM) and the Society of Critical Care Medicine (SCCM). EVIDENCE: The task force members provided the personal experience and determined the published literature (MEDLINE articles, textbooks, pharmacopeias, etc.) from which consensus would be sought. Published literature was reviewed and classified into one of four predetermined categories, according to study design and scientific value. CONSENSUS PROCESS: The task force met several times as a whole, and numerous times in smaller groups by teleconference, over a 1-yr period to identify the pertinent literature and arrive at consensus recommendations for the whole task force to discuss. Consideration was given to the relationship between the weight of scientific information and the experts' viewpoints. Over the next year, draft documents were composed by a task force steering committee and debated by the task force members until consensus was reached by nominal group process. The task force draft was then reviewed, assessed, and edited by the Board of Regents of the ACCM. After steering committee approval, the draft document was reviewed and approved by the SCCM Council. DATA SYNTHESIS: To facilitate rapid communication of the three recommendations contained within the complete and unabridged practice parameter document, an executive summary was prepared for publication by the ACCM Board of Regents, and this executive summary was approved by the task force steering committee and the SCCM Executive Council. CONCLUSIONS: A consensus of experts provided three recommendations with supporting data for achieving sustained neuromuscular blockade in critically ill patients: a) pancuronium is the preferred neuromuscular blocking agent for most critically ill patients; b) vecuronium is the preferred neuromuscular blocking agent for those patients with cardiac disease or hemodynamic instability in whom tachycardia may be deleterious; c) patients receiving neuromuscular blocking agents should be appropriately assessed for the degree of blockade that is being sustained. This executive summary selectively presents supporting information and is not intended as a substitute for the complete document.

Adult↗
PubMed1994

Soluble guanylate cyclase from bovine lung: activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous and ferric states.

Nitric oxide (.NO) is a recently discovered signaling agent which plays a role in many biological processes such as vasodilation and neuronal synaptic transmission. The only receptor characterized thus far for .NO is the soluble form of guanylate cyclase (sGC). .NO increases the Vmax of sGC by 100-200-fold, probably by interacting with a heme moiety on the enzyme. Although several procedures exist for purifying sGC, these procedures result in preparations with low heme contents. Using a novel procedure, the enzyme has been purified to homogeneity from bovine lung with a heme content of approximately 1 heme/heterodimer. The UV-visible spectrum of the enzyme contains a Soret peak centered at 431 nm and a single broad alpha/beta peak at 555 nm indicative of a 5-coordinate ferrous heme with histidine as the axial ligand. The heme moiety does not bind oxygen but will readily bind .NO to form a 5-coordinate complex or carbon monoxide (CO) to form a 6-coordinate complex. Oxidation of the heme with ferricyanide shifts the Soret to 393 nm, due most likely to the formation of a 5-coordinate ferric heme. In the ferric state, the heme will apparently not bind water but will bind cyanide with reduced affinity compared to methemoglobin and metmyoglobin. Purified enzyme containing 1 heme/heterodimer is activated 130-fold by .NO and 4.4-fold by CO.

Animals↗
PubMed1992

Evaluation of the standardized disk diffusion and agar dilution antibiotic susceptibility test methods by using strains of Neisseria gonorrhoeae from the United States and Southeast Asia.

Presently, most Neisseria gonorrhoeae testing is based on beta-lactamase tests and agar dilution with common therapeutic agents. The National Committee for Clinical Laboratory Standards (NCCLS) recently described a disk diffusion test that produced results similar to the reference agar dilution method for the antibiotic susceptibility of N. gonorrhoeae. We obtained 71 gonococcal isolates from active-duty males aboard a United States Navy vessel while deployed in the Western Pacific during 1989. In addition, 47 isolates of N. gonorrhoeae were obtained from sexually transmitted disease clinics within the branch clinic operations of the Naval Hospital, San Diego (SD), and tested. Antibiotic susceptibility tests by using the NCCLS agar dilution and disk diffusion techniques were compared. Among the Southeast Asia (SEA) isolates, 47% were beta-lactamase producers compared with 10.5% of the SD isolates. The mean MICs (SEA/SD) in micrograms per milliliter for both groups were as follows: penicillin, 88/15; tetracycline, 2.2/0.95; erythromycin, 1.2/0.49; ceftriaxone, 0.016/0.012; cefotaxime, 0.034/0.03; cefuroxime, 0.44/0.17; cefoxitin, 1.3/0.97; spectinomycin, 150/131; ciprofloxacin, 0.07/0.034; norfloxacin, 0.77/0.29; lomefloxacin, 0.15/0.0.056; and ofloxacin, 0.07/0.036. The established NCCLS interpretive criteria for both susceptibility methods appear applicable to domestic gonococcal strains. However, modifications may be necessary for the more antimicrobial agent-resistant SEA isolates on the basis of the clinical success and cure rates following the indicated single-dose regimens for the geographic region.

Asia, Southeastern↗
PubMed1991

Reconstitution of catalytically competent human zeta-thrombin by combination of zeta-thrombin residues A1-36 and B1-148 and an Escherichia coli expressed polypeptide corresponding to zeta-thrombin residues B149-259.

Human zeta-thrombin, a catalytically competent serine proteinase, arises from a single chymotryptic cleavage at Trp-148 in alpha-thrombin to generate two nonconvalently associated polypeptide segments designated zeta 1-thrombin (the 36-residue A-chain disulfide linked to B-chain residues B1-148) and zeta 2-thrombin (B149-259). We report here the expression of recombinant zeta 2-thrombin in Escherichia coli and the reconstitution of catalytically competent zeta-thrombin by combination of zeta 1-thrombin with recombinant zeta 2-thrombin. A DNA fragment encoding zeta 2-thrombin was cloned into a pATH2 expression vector as a trpE-zeta 2 fusion gene, in which a factor Xa cleavage site was inserted between the trpE and the zeta 2-thrombin gene. High-level expression of this fusion protein was achieved under the control of the E. coli trp promoter. The expressed zeta 2-thrombin was liberated from the fusion protein by factor Xa cleavage, reduced with DTT, and purified to homogeneity by reverse-phase HPLC. Oxidation of the reduced zeta 2-thrombin in the presence of 80 microM CuSO4 and 6 M urea at pH 8.15 yielded material that was indistinguishable on HPLC from zeta 2-thrombin isolated by resolution of human zeta-thrombin. Catalytically active zeta-thrombin was generated by combination of recombinant zeta 2-thrombin with zeta 1-thrombin that was isolated by resolution of human zeta-thrombin. Recombinant zeta-thrombin displayed catalytic activities, toward a small chromogenic substrate and fibrinogen, that were similar to those of alpha-thrombin prepared from human blood plasma and zeta-thrombin obtained by treatment of alpha-thrombin with chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Assessing phonology in young children.

We have presented a discussion of three important concepts regarding early phonological assessment. The first is that the child's language level must be considered in collecting and analyzing a sample and in interpreting the results. For a child with age-level language abilities, the sample should consist of both a single-word test and a running speech sample; both independent and relational analyses are appropriate. For a child with delayed expressive language and a small productive vocabulary, a sample comprised of spontaneous productions is more appropriate. In this case, the sample should be analyzed in terms of sound classes and syllable and word shapes that occur; a phonological process analysis is inappropriate. Lexical selection patterns should be noted. The results of the analyses should be interpreted in view of the expectations for the child's language level. A child with a normally developing language system is expected to have more advanced phonology than a child of the same age with delayed language. Thus, a child with a large vocabulary and word combinations is expected to have an expanding phonological system, with a full range of sound classes and syllable and word shapes. If a child is delayed in language and is still within the first 50-word stage, the expectation is that the phonological system will be more limited. Critical features for the phonology of early productive vocabulary have been identified. Lack of one or more of these features is indicative of atypical phonological development at any age and language level. The second concept is that the phonological system as a whole must be considered. In particular, the analyses and expectations should be based on the presence or absence of sound classes and syllable shapes rather than on sounds per se. Lack of an entire class or syllable structure would be cause for concern; lack of a particular sound, even though it has been shown to be acquired early, would not be. Thus, lack of the entire fricative class at 36 months would be of concern, whereas errors on /f/, which according to Prather et al., 1975, is mastered by this age, would not be. The third important concept emerges from the case studies; both studies demonstrate that longitudinal assessment is necessary to document changing profiles over the course of development. A child such as David, who has a normal-but-delayed profile in language and phonology at one age, may subsequently exhibit atypical patterns as phonology and language dissociate.(ABSTRACT TRUNCATED AT 400 WORDS)

Articulation Disorders↗
PubMed1983

In vitro antimicrobial activity of eight new beta-lactam antibiotics against penicillin-resistant Neisseria gonorrhoeae.

Increasing numbers of cases of penicillin-resistant gonorrhea necessitate the evaluation of new antibiotics for treatment of this disease. We tested the susceptibility of 92 penicillinase-producing (PP) Neisseria gonorrhoeae isolates and 88 penicillin-susceptible (PS) isolates to eight new beta-lactam antibiotics. The minimal inhibitory concentrations of these antibiotics were determined by the agar plate method. PP and PS N. gonorrhoeae isolates were susceptible to clinically achievable levels of all antibiotics tested. There were, however, marked differences among the drugs with regard to the concentration required to inhibit growth. The PP N. gonorrhoeae isolates were extremely susceptible to ceftriaxone, ceftizoxime, and cefotaxime, highly susceptible to moxalactam and cefoperazone, and less susceptible to cefoxitin, ceforanide, and cefonicid (geometric mean minimal inhibitory concentrations were 0.002, 0.003, 0.007, 0.03, 0.07, 0.6, 2.4, and 3.1 micrograms/ml, respectively). Although this in vitro study showed PP N. gonorrhoeae isolates to be comparatively more susceptible to ceftriaxone, ceftizoxime, and cefotaxime than to the other antibiotics, these results may not correlate with clinical efficacy.

Anti-Bacterial Agents↗
PubMed1979

Platelet-associated immunoglobulin G in childhood idiopathic thrombocytopenic purpura.

Childhood ITP is an acquired hemorrhagic disorder with a heterogeneous clinical course. We measured PAIgG levels in 20 children with ITP (7 acute, 13 chronic). Both groups had significantly greater PAIgG values than age-matched normal subjects and thrombocytopenic controls (P less than 0.001). In addition, PAIgG values in chronic ITP were significantly lower than those in acute ITP (P less than 0.003). Serial PAIgG values were obtained in some patients; most returned to normal in association with clinical recovery. The measurement of PAIgG is useful in the diagnosis and follow-up of childhood ITP. PAIgG values may assist in differentiating acute and chronic disease in children.

Acute Disease↗