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J Shaffer

Publications and source records attributed to J Shaffer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Detection of conformational changes along the kinetic pathway of protein kinase A using a catalytic trapping technique.

The dissociation rate constants for the two products of the reaction catalyzed by protein kinase A, ADP and phosphopeptide, were measured using a catalytic trapping technique to determine the role of product release in enzyme turnover. The enzyme was preequilibrated with ADP, and the reaction was initiated with a peptide substrate, LRRASLG, and ATP in a rapid quench flow instrument. At high, free magnesium concentrations (>2 mM), the large 'burst' in phosphopeptide production disappears, and, at low concentrations of free magnesium (0.5-1 mM), the kinetic transients become sigmoidal prior to the linear turnover phase. Increasing the concentrations of ATP or ADP did not influence the shape of the kinetic transients in the first 20 ms. ADP preequilibration protects the enzyme from inhibition by the covalent inactivator p-fluorosulfonylbenzoyl 5'-adenosine at 0.5 mM free magnesium, indicating that a competent E. ADP complex forms at low metal concentrations and the sigmoidal behavior in the catalytic trapping experiment is not due to free enzyme at high ATP concentrations. Simulations of the data indicate that ADP release is rate-limiting for turnover at high magnesium concentrations, but, at lower physiological levels of 0.5 and 1 mM, the off rate of ADP is 3- and 2-fold higher than k(cat), respectively. In contrast, the initial portions of the kinetic transients at 0.5 mM free magnesium were unaffected by phosphopeptide preequilibration, indicating that the release rate of this product is significantly larger than turnover. The transient kinetic data, coupled with a previous report [Shaffer and Adams (1999) Biochemistry 38, 5572-5581], support a phosphorylation mechanism under physiological magnesium concentrations that incorporates two partially rate-determining conformational changes, one prior to and one after the phosphoryl transfer step. We propose that the initial step activates the enzyme through key positioning of one or more active-site residues and the second step relaxes this conformation, a prerequisite for a subsequent catalytic cycle.

Adenosine↗
PubMed1999

An ATP-linked structural change in protein kinase A precedes phosphoryl transfer under physiological magnesium concentrations.

The kinetic mechanism for the catalytic subunit of protein kinase A was evaluated using physiological concentrations of free magnesium (0.5 mM) and a rapid quench flow technique. When the enzyme is pre-equilibrated with ATP, the peptide substrate, LRRASLG (Kemptide), is phosphorylated in a biphasic manner with a rapid, exponential "burst" phase (kb) followed by a slower, linear phase (kL) that corresponds to the steady-state kinetic rate. Both the amplitude and the substrate-rate dependence of the initial, burst phase indicate that the rate of phosphoryl transfer is fast (approximately 500 s-1) and does not limit turnover (45 s-1). Viscosity studies indicate that, while Kemptide is in rapid equilibrium, ATP does not exchange rapidly with the active site and kcat/KATP is limited by the rate constant for nucleotide encounter. When the pre-steady-state kinetic experiments are initiated with ATP, a lag phase is observed at low ATP concentrations consistent with rate-limiting association. At high ATP concentrations (>1 mM), a burst phase is observed but the rate and amplitude are low on the basis of the bimolecular rate constant for ATP association and the rate constant for phosphoryl transfer. The kinetic data indicate that the phosphoryl transfer step is fast at physiological magnesium concentrations, but an ATP-linked conformational change precedes this step, limiting the burst phase rate constant. Simulations of the pre-steady-state kinetic transients indicate that turnover (45 s-1) is limited both by net product release (70 s-1) and by this structural change (170 s-1). This structural change may also occur at high free magnesium concentrations, but it must be significantly faster than 170 s-1 and, consequently, not rate-limiting for turnover (kcat = 20 s-1 at 10 mM free Mg2+). We propose that this conformational event is an obligatory component of the kinetic pathway and includes a movement of the catalytic residues necessary for supporting phosphoryl group donation.

Adenosine Triphosphate↗
PubMed1999

Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library.

A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5-1-mm layer of agarose containing 130 micron diameter TentaGel beads at a density of 2-20 beads/mm2. Employing this technique a 9-mer, 442,368-member peptide library (designed around the 13 amino acid alpha-MSH peptide sequence) made up of 12 separate pools of 36,864 peptides/pool was assayed. Initially, a fraction (approximately 10%) of each pool was scanned (approximately 3700 beads from each pool) in 60-mm petri dishes to identify the most active pools. Upon direct photocleavage of the beads with UV light (365 nm), each petri dish was photographed over a 60-min period with a CCD camera to record changes in light intensity as an index of melanosome dispersion. Active beads were those that were surrounded by a localized decrease in light transmittance indicating melanosome dispersed cells. Upon examination with a dissecting microscope, single beads centrally located to a circular array of dispersed cells were identified and removed from the agarose and sequenced by Edman degradation to determine the peptide sequence. Re-synthesized peptides were re-examined against alpha-MSH receptor to confirm and quantify the activity. Several 9-mer peptides were identified with potencies similar to the natural 13-mer peptide. This method allows for the rapid screening of large bead-based photo-cleavable peptide libraries with the advantage that each compound is screened as a discrete molecule in a well-less format.

Animals↗
PubMed1999

Home parenteral nutrition in adults: a european multicentre survey in 1997. ESPEN-Home Artificial Nutrition Working Group.

A retrospective survey on home parenteral nutrition (HPN) in Europe was performed from January to December 1997. Data were compared to a similar study performed in 1993. A questionnaire of HPN practice was designed by the members of the ESPEN-HAN group. This involved adult patients (older than 16 years) newly registered in an HPN program between 1 January and 31 December 1997 and included: number of patients, underlying diseases and a 6-12 month outcome. Incidence and prevalence (at 1.1.1998) of adult HPN were calculated according to the estimated total population in 1997 for the countries in which more than 80% of HPN patients were reported.A total of 494 patients were registered in 73 centres from nine countries (Belgium (B), Denmark (D), France (F), Poland (P), Spain (S), Sweden (Sw), United Kingdom (UK), The Netherlands (N) and Germany (G). The underlying diseases for HPN in 494 patients were cancer (39%), Crohn's (19%), vascular diseases (15%), radiation enteritis (7%), AIDS (2%), other diseases with intestinal failure (18%). Incidence (patients/million inhabitants/year) were in N (3), F. (2.9), D. (2.8), B. (2.6), UK (1. 2), S (0.7) and P (0.36), respectively. Prevalence were in D. (12.7). U.K. (3.7), N. (3.7), F (3.6), B (3.0), P (1.1), S (0.65). After this 6-12 months follow-up (n=284), the mortality was respectively 4% in Crohn's disease, 13% in vascular diseases, 16% in others, 21% in radiaton enteritis, 34% in AIDS, 74% in cancer. Incidences and prevalences modestly increased in these seven European countries in 1997 in comparison to 1993. The percentages of underlying diseases in these countries remained similar except for AIDS that significantly decreased (from 7% to 2%). Outcomes did not significantly differ in the 4-year period except for AIDS (34% instead of 88% mortality) and could have been related to newer, more efficacious therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Tartrate-resistant acid phosphatase forms complexes with alpha2-macroglobulin in serum.

Tartrate-resistant acid phosphatase (TRAP) is a standard histochemical marker of differentiated osteoclasts and has been proposed as a serum/plasma marker for osteoclast activity. Enzyme assays have been described that show elevated TRAP enzyme activity in the serum or plasma of patient groups known to have increased bone metabolism. However, the poor stability of the enzyme and potential contribution from nonosteoclastic sources make it problematic to measure in patient samples. Immunoassays developed to measure TRAP in serum and plasma have yielded widely varying TRAP levels in both normal and disease states. It is not clear if this variability is caused by differences in assay calibration, antibody specificity, and/or TRAP instability. In this paper, we report that purified TRAP spiked into serum forms high molecular weight complexes. Complex formation results in greatly decreased TRAP enzyme activity and immunoreactivity. The complexing protein in serum has been identified as alpha2-macroglobulin (alpha2M). Similar complexes are observed in stored patient samples. In vitro studies with purified components show that TRAP binds to alpha2M primarily through noncovalent ionic interactions. Our results demonstrate that one mechanism of TRAP instability in serum is complex formation with alpha2M and suggest further that current TRAP enzyme and immunoassays may not accurately measure the circulating level of TRAP.

Acid Phosphatase↗
PubMed1998

Patients with stable chronic obstructive pulmonary disease can safely undergo intravenous dipyridamole thallium-201 imaging.

BACKGROUND: Patients with chronic obstructive pulmonary disease are usually excluded from intravenous dipyridamole thallium-201 testing. We developed a nurse-administered protocol to screen and pretreat patients so they could be safely tested. METHODS AND RESULTS: We prospectively screened patients referred for intravenous dipyridamole thallium testing and retrospectively reviewed a comparison group of patients who had undergone intravenous dipyridamole testing before our bronchospasm protocol. We studied 492 consecutive patients referred for intravenous dipyridamole thallium testing, separating those with complete data (n = 451) into two groups: group A (n = 72), patients assessed to be at risk for intravenous dipyridamole-induced bronchospasm who received our bronchospasm treatment protocol; and group B (n = 379), patients assessed to be free of risk, who did not receive our bronchospasm protocol. Group C (n = 89) was a retrospective comparison group of patients who had undergone intravenous dipyridamole testing before initiation of the protocol. Patients were considered at risk for an adverse event if any of the following were present: peak flow < or =400 ml at the time of the test (spirometry by nurse) that increased to >400 ml after bronchodilator treatment, wheezing audible with stethoscope, history of chronic obstructive pulmonary disease or asthma or dyspnea on exertion at less than four blocks, or resting respiratory rate >18 breaths/min. The test was considered contraindicated if resting oxygen saturation was <85%, respiratory rate < or =36 breaths/min, or peak flow measured by peak flowmeter <400 ml after bronchodilator inhalant (albuterol or metaproterenol sulfate by spacer) at a dose of up to six puffs. One minute after injections of thallium-201, patients at risk were given 50 mg aminophylline by slow intravenous injection. We looked for major and minor adverse effects and divided them into three categories: (1) minor events (transient headache, abdominal discomfort, or nausea), wheezing (audible by stethoscope but without marked respiratory distress), (2) marked events (severe bronchospasm or severe ischemia defined as wheezing audible with or without stethoscope, respiratory rate >20 breaths/min or increased by 10 from pretest evaluation, oxygen desaturation to <90%, hypoventilation [reduced respiratory rate with decreased mental status], respiratory arrest, chest pain, horizontal ST-segment depression > or =1 mm on the electrocardiogram in any lead, symptomatic hypotension), or (3) other intravenous dipyridamole-induced side effects (persistent headache, dizziness, flushing, nausea, dyspnea, and ischemic chest pain) or anginal equivalent. The protocol properly identified patients with impaired pulmonary function. There was no difference in the frequency of adverse marked events among groups A, B, or C (1 % vs 4% vs 2%, p = 0.25). Patients in group A had more minor side effects than those in group B (53% vs 35%, p = 0.004). Specifically, patients in group A were more likely to wheeze (39% vs 1 %, p = <0.001), but wheezing in group A was self-limited or responded to treatment as described in the protocol. The prevalence of positive thallium-201 scans in group A (44%) compared with group C (49%) was not different (p = 0.15). CONCLUSIONS: A nurse-administered risk assessment and pretreatment protocol (1) properly identified patients with impaired pulmonary function, (2) permitted completion of intravenous dipyridamole testing in patients at risk for bronchospasm without an increased incidence of marked adverse events, and (3) did not appear to influence the interpretation of the thallium test.

Aged↗
PubMed1996

Properties of beta-lactamase from Pseudomonas syringae.

Pseudomonas syringae isolate BR2R produces tabtoxin, a beta-lactam-containing antibiotic, and the causative agent of wildfire disease of green bean (Phaseolus vulgaris). beta-Lactamase production has been suggested as the mechanism that protects P. syringae from tabtoxin. We sought to determine whether the organism produces beta-lactamase and whether the enzyme plays a role in protection from this antibiotic. P. syringae and mutants defective in tabtoxin production and resistance produce beta-lactamase. Three distinct beta-lactamases with molecular weights of 41,000 were identified. The isoelectric points of the proteins were 6.1, 6.8, and 9.2. The enzymes preferentially hydrolyze cephalosporin. This investigation demonstrates that the organism produces multiple beta-lactamases and describes characteristics of the proteins.

Isoelectric Focusing↗
PubMed1996

The effect of drug therapy on long-term outcome of childhood asthma: a possible preview of the international guidelines.

BACKGROUND: International guidelines classify childhood asthma as mild, moderate, and severe and recommend treatment with "as needed" bronchodilators, cromolyn sodium, and inhaled corticosteroids, respectively. Recently, some investigators proposed inhaled corticosteroids as first-line therapy to avoid possible irreversible airway obstruction. This article describes a retrospective study assessing the effect of the guidelines' approach on the long-term outcome of childhood asthma and the possible effect of delaying initiation of administration of corticosteroids. METHODS: A retrospective review was performed on the charts of 175 children, and an end-of-study questionnaire plus results of spirometry completed the data. The mean age at start of study was 6.5 years, and the children were followed up for 2.2 to 16.8 years (mean, 8.4 years). Treatments included bronchodilators as needed only (patients with mild asthma), cromolyn sodium (patients with moderate asthma), and inhaled corticosteroids (patients with severe asthma). RESULTS: Frequency of symptoms, unscheduled doctors' visits, emergency department visits, hospitalizations, and missed school days decreased for the whole group. Statistically significant between-treatment differences favored both antiinflammatory therapies over as-needed bronchodilator treatment (cromolyn sodium: symptoms, p < 0.05; hospitalizations, p < 0.05; corticosteroids: emergency department visits, p < 0.05; hospitalizations, p < 0.05). Mean spirometry results improved, and the postbronchodilator values approached normal by study end. Assessed by treatment, spirometry decreased with bronchodilators but improved in patients treated with cromolyn sodium or inhaled corticosteroids. Delay in starting administration of cromolyn sodium had an unfavorable effect on clinical outcomes (p < 0.01) and spirometry (p < 0.05); delay in starting administration of corticosteroids did not. CONCLUSIONS: Treatment with antiinflammatory drugs (cromolyn sodium or inhaled corticosteroids), but not as-needed bronchodilators alone, improves the long-term prognosis of asthma. It is possible that starting administration of nonsteroid antiinflammatory agents earlier than the present recommendations could further improve clinical outcomes, but prospective studies are needed.

Adolescent↗
PubMed1996

Home parenteral nutrition in adults: a multicentre survey in Europe in 1993.

A retrospective survey was performed in 1994, involving 496 adult home parenteral nutrition (HPN) cases, newly enrolled in the year 1993 from 13 European countries from 75 centres. From the 8 countries having registered more than 80% of cases (423 patients), incidence and prevalence ranged from 0.2 to 4.6 and 0.3 to 12.2 patients/10(6) population/year. In the patients studied, the diagnosis was cancer (42%), Crohn's disease (15%), vascular diseases (13%), radiation enteritis (8%), AIDS (4%) and other nonmalignant non-AIDS diseases (18%). Short bowel syndrome and intestinal obstruction were the two major indications for HPN in 31% and 22%, respectively. Seventy-three percent of the centres had a nutrition team. HPN was administered through a tunnelled venous central catheter in 73%, cyclical nocturnal infusions were used in 90% of patients, and intravenous feeding was the sole source of nutrition in 33%. Only 44% undertook HPN unaided. The present report indicates that cancer has now become the main indication for HPN in Europe; there was, however, a heterogeneous distribution of diseases amongst the reporting countries. The observed 9 (6-12)-month probability of survival was poor in AIDS (n = 8; 12%) and cancer patients (n = 78; 29%) but better for the other HPN indications (n = 115; 92%).

Journal Article↗
PubMed1996

Long-term glucose homeostasis in patients on home parenteral nutrition.

Glycated haemoglobin (Hb A1c) was used as a marker of long-term hyperglycaemia in patients receiving high glucose loads for home parenteral nutrition (HPN). Thirty-six patients received a median weekly glucose delivery of 2000 g (800-4200) at a median infusion rate of 11.8 mg/kg/min (5.0-19.2). There was no correlation between Hb A1c (median- 3.1%, range 2.0-6.3%) and glucose infusion rate or weekly glucose load. It is concluded that significant prolonged hyperglycaemia does not appear to be a problem in patients on HPN managed by this unit.

Journal Article↗
PubMed1995

Reducing low-density lipoprotein cholesterol levels in an ambulatory care system. Results of a multidisciplinary collaborative practice lipid clinic compared with traditional physician-based care.

BACKGROUND: The Cincinnati (Ohio) Department of Veterans Affairs Medical Center Lipid Clinic was established as a collaborative practice to treat patients with substantially elevated serum cholesterol levels referred from the General Internal Medicine Clinic. The Lipid Clinic team (led by a clinical nurse), included a clinical pharmacist, nurse practitioner, dietitian, and clinical psychologist. A consultant cardiologist reviewed all laboratory tests and confirmed therapeutic decisions at a weekly preclinic meeting. OBJECTIVE: To compare the success of a limited term of treatment in the Lipid Clinic with that of standard physician-based care in the General Internal Medicine Clinic in achieving the goals recommended by the National Cholesterol Education Program 1 for low-density lipoprotein cholesterol. METHODS: A convenience sample of age-matched patients with total cholesterol levels greater than 6.85 mmol/L (265 mg/dL) was selected from each clinic (Lipid Clinic, n = 60; General Internal Medicine Clinic, n = 60). Fasting lipid profiles were drawn in the free-living state and in the sitting position, and matched by month. Treatment of patients in the Lipid Clinic group consisted of evaluation and treatment of secondary causes of hyperlipidemia, goal setting, and treatment according to the National Cholesterol Education Program I algorithm. Counseling and education were individualized. Outcomes were determined after four visits (12 and 18 months for the Lipid Clinic and General Internal Medicine Clinic groups, respectively). Patients in the two groups had comparable risk factors, including presence of coronary heart disease. RESULTS: After four clinic visits, patients in the Lipid Clinic group were four times more likely to reach a National Cholesterol Education Program I goal of a low-density lipoprotein cholesterol level less than 3.36 mmol/L (130 mg/dL) than were comparable patients in the General Internal Medicine Clinic group (relative risk, 4.1; 95% confidence interval, 1.4 to 12.7; P < .001). CONCLUSION: These results support multidisciplinary, goal-oriented collaborative practice as an efficacious model of preventive medicine and health care provision.

Aged↗
PubMed1995

Short-term and long-term effects of albuterol aerosol therapy in cystic fibrosis: a preliminary report.

The effectiveness of maintenance albuterol aerosol therapy in cystic fibrosis (CF) was assessed by comparing spirometric measurements at the beginning and end of 1 year. Peak expiratory flow rates (PEFR) were measured twice daily to determine bronchodilator responsiveness and spontaneous diurnal variation (SDV), and results were compared with groups of normal and asthmatic children. CF patients not receiving regular albuterol therapy served as a control group. In the treatment group, forced vital capacity (FVC) and forced expiratory volume in the first second (FEV1) were significantly increased (12.2% and 18.4%, respectively) over the course of the treatment year, as contrasted with a significant decrease during the preceding year. During the study year the CF control group had a significant decrease in FEV1 and FEF25-75%, and the difference between treatment and control groups was significant for FVC, FEV1, and FEF25-75%. PEFR increased from the first to the last week of the year-long observation period (from 71.8% to 78.7% of predicted values, P < 0.01). Spontaneous diurnal variations were significantly greater in the CF study group than a group of normal children; SDV decreased significantly in the treatment group during the year of study. A bronchodilator response of > 15% was present in 25.8% of CF patient days, but there was considerable interpatient variability. Frequent bronchodilator responders were accurately predicted by their baseline bronchodilator responsiveness, but not by age or personal or family history of asthma or atopy. No difference in long-term pulmonary function improvements were noted between frequent and infrequent responders. The results suggest that maintenance albuterol aerosol treatments reversed the progressive downward course in lung function in the CF treatment group. A double-blind placebo-controlled study is required to confirm these preliminary findings.

Administration, Inhalation↗
PubMed1995

Review article: artificial nutritional support for improved patient care.

Malnutrition is common and undiagnosed in the majority of affected hospital patients; it is associated with impaired organ function, morbidity, and increased length of hospital stay. Artificial nutritional support in malnourished patients leads to improvement in nutritional status and clinical outcome. Nutritional support is required in malnourished patients, patients who are unable to take normal diet and patients with intestinal failure. Gastroenterologists are required to supervise patients with intestinal failure, to insert endoscopic feeding devices, and increasingly to participate in, or lead, nutritional support teams. Major developments in nutrient delivery have included percutaneous endoscopic feeding devices, the recognition that enteral feeding is possible in patients with gastric stasis, and that nutrient needs can be met by peripheral parenteral nutrition. There is much interest in the use of new substrates, or substrates delivered in pharmacological doses such as glutamine and arginine, to manipulate the response to disease. Many hospitals lack an organized approach to artificial nutritional support. Patients continue to suffer from a lack of treatment or the consequences of inappropriate or inadequate treatment. This article reviews the current status of artificial nutritional support and provides guidelines for patient management.

Enteral Nutrition↗
PubMed1995

Treatment of perilunate and transscaphoid perilunate dislocations of the wrist.

Dorsal perilunate dislocations (PLDs) and dorsal transscaphoid perilunate dislocations (TS-PLDs) are reviewed in this paper, including the diagnosis, initial treatment, and options for definitive treatment. Closed reduction to obtain gross carpal alignment is performed on an urgent basis. Anatomic reduction is essential and is most consistently obtained by open-reduction, percutaneous pin fixation. A dorsal approach alone provides good visualization of the radiocarpal and midcarpal joints and allows efficient stabilization of dislocations and fracture-dislocations. To achieve stable fixation of the carpal bones, the senior author (JS) has developed a surgical technique that employs a specific order and configuration of Kirschner-wire placement. The stabilized joints parallel the major ligaments disrupted in PLD and TS-PLD: the volar radiocapitate, radiotriquetral, and radioscaphoid ligaments. Maintenance of stable anatomic reduction of the radiocarpal and midcarpal joints during healing is the key to satisfactory early and long-term clinical results.

Adult↗
PubMed1994

Cyclosporin A does not affect the absolute rate of cortical bone resorption at the organ level in the growing rat.

The weanling rat, an animal model of rapid bone turnover, was used to evaluate the effects of various doses of cyclosporin A (CsA) on various bones during different time periods. Sprague-Dawley male rats were extensively prelabeled with 3H-tetracycline during 1-3 weeks of age. At 4 weeks of age, four groups of rats were given daily subcutaneous injections: vehicle or CsA--low dose (10 mg/kg), intermediary dose (20 mg/kg), or high dose (30 mg/kg) for 7, 14, or 28 days. Three different whole bones--the femur (low turnover), scapula (moderate turnover), and lumbar-6 vertebra (high turnover) were harvested intact at 4, 5, 6, and 8 weeks of age. The whole bones were assayed weekly for total dry defatted weight, calcium mass (formation), and loss of 3H-tetracycline (bone resorption) following treatment with CsA. Serum CsA levels, calcium creatinine, and alkaline phosphatase were measured weekly. Significant decreases in serum calcium and alkaline phosphatase were observed at 1 and 2 weeks, and were normalized by 4 weeks of treatment. No significant changes in serum creatinine were noted. For all three doses of CsA, no effect was observed on the absolute rate of cortical bone resorption of three different, whole bones over three time periods. Body weight and bone formation in treated animals was significantly smaller in a dose- and time-related fashion compared with control animals at sacrifice. However, compared with the initial control animals, body weights and bone masses of the final treated animals were much larger, suggesting that the smaller bone masses were due to insufficient growth and slow gain in bone mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗