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Biomedical subjects

J A Adams

Publications and source records attributed to J A Adams.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cellular immune responses to four doses of percutaneous bacille Calmette-Guérin in healthy adults.

To explore the hypothesis that low-dose immunization might induce preferential Th1 cell immunity, 76 adults were vaccinated with one of four doses of bacille Calmette-Guérin (BCG): The doses contained very low (1.6 x 10(5) cfu), low (3.2 x 10(6) cfu), standard (1.6 x 10(8) cfu), or high (3.2 x 10(8) cfu) levels of BCG. Delayed-type hypersensitivity responses occurred 8 weeks after vaccination in 10% of persons given very low or low doses of BCG, compared with 95% and 100% of persons given standard or high doses, respectively. Lymphoproliferative responses, which were increased only for high-dose vaccinees, peaked 2 weeks after vaccination and were directed chiefly against Mycobacterium tuberculosis-secreted proteins, particularly the antigen 85 complex. Significant increases in mycobacteria-specific interferon-gamma expression were present 16 weeks after vaccination only for persons given standard or high doses of BCG. Percutaneous BCG appears capable of inducing a temporary Th1-like immune response, but standard or higher dosages are required.

Administration, Cutaneous↗
PubMed1997

Participation of ADP dissociation in the rate-determining step in cAMP-dependent protein kinase.

Pre-steady-state kinetic analyses of the catalytic subunit of cAMP-dependent protein kinase showed that the rate constant for phosphoryl transfer is fast and either the release of one or both of the products or a conformational change controls turnover [Grant, B., & Adams, J. A. (1996) Biochemistry 35, 2022-2029]. To determine which step or steps control turnover in the wild-type enzyme, we used a catalytic trapping technique to measure directly the dissociation rate constant for ADP. The phosphorylation of two peptide substrates, LRRASLG and GRTGRRNSI, was monitored using a rapid quench flow technique under conditions where saturating concentrations of ADP were preequilibrated with the enzyme before excess ATP and one of the substrates were added to trap the free enzyme and to start the phosphorylation reaction. Under ADP preequilibration conditions, no 'burst' phase was observed, and although the rate of linear, steady-state turnover was unaffected, the net production of phosphopeptide lagged behind the non-preequilibrated control. This phenomenon occurs due to the slow release of the product, and kinetic modeling suggests that this effect can be explained if the dissociation rate constant for ADP is 24 s-1 and solely limits turnover (kcat = 23 s-1) for the phosphorylation of LRRASLG. Using GRTGRRNSI, the dissociation rate constant for ADP is 35 s-1 and limits turnover (kcat = 29 s-1) if the reaction is initiated by the addition of enzyme. Under preequilibration conditions with either ATP or GRTGRRNSI, turnover is approximately 50% lower, suggesting that ADP release may partially control this parameter. This preequilibration effect can be explained by slowly interconverting enzyme forms with specific peptide-induced turnover properties. These studies indicate that ADP release is an essential rate-limiting component for turnover but also suggests that other factors contribute subtly when the structure of the substrate is altered.

Adenosine Diphosphate↗
PubMed1997

Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

Amino Acid Sequence↗
PubMed1997

Identification of a partially rate-determining step in the catalytic mechanism of cAMP-dependent protein kinase: a transient kinetic study using stopped-flow fluorescence spectroscopy.

The kinetics of nucleotide binding and phosphoryl group transfer were measured in the catalytic subunit of cAMP-dependent protein kinase using stopped-flow fluorescence spectroscopy and an acrylodan-labeled derivative of this enzyme, which we have previously shown to have kinetic properties similar to those for the wild-type enzyme (Lew et al., 1996). The fluorescence emission spectrum of this enzyme is quenched differentially by ATP and ADP so that both the binding of ligands and phosphoryl group transfer at the active site can be monitored selectively. The association and dissociation rate constants for both nucleotides were measured using two methods: relaxation and competition binding. The ratio of the observed dissociation and association rate constants by both methods are consistent with Kd measurements (25 microM) determined by equilibrium fluorescence quenching. The dissociation rate constant for ADP (100 s(-1)) is approximately 2.5-fold larger than k(cat) (39 s(-1)). A full viscosity effect was measured for k(cat), suggesting that a diffusive step or steps limit maximum turnover. Pre-steady-state kinetic transients are biphasic and were fitted to observed rate constants of 500 s(-1) and 60 s(-1) at 500 microM Kemptide (LRRASLG). Metal substitution studies (Mg2+ vs Mn2+) indicate that this first phase represents the phosphoryl group transfer step. Phosphopeptide release is faster than this second phase since the substrate is in rapid exchange with the enzyme and phosphorylation reduces the affinity of the peptide. The inability to assign this second phase to the chemical event or to product release implies that it reflects a viscosity-sensitive, protein conformational change that occurs after phosphoryl group transfer and prior to product release. Two conformational steps were detected in the binding of both ATP and ADP by relaxation methods that may be related to this second pre-steady-state kinetic phase. We suggest that this additional step in the kinetic mechanism may also occur in the wild-type enzyme and represents a large structural change in the enzyme during normal catalytic cycling.

Adenosine Diphosphate↗
PubMed1997

Is there a catalytic base in the active site of cAMP-dependent protein kinase?

The carboxyl group of an aspartic acid in the active site of the serine-specific protein kinase, cAMP-dependent protein kinase, is poised near the hydroxyl proton of a peptide substrate in the X-ray crystallographic structure (Madhusudan et al., 1994), suggesting that this residue may act as a general-base catalyst in the phosphoryl transfer reaction. Indeed, several proposals have been made in this regard. We measured the pre-steady-state kinetics in this enzyme using a rapid quench flow technique to understand the role of this putative base. The phosphorylation of the peptide substrate, GRTGRRNSI, by cAMP-dependent protein kinase exhibited "burst" kinetics consistent with a mechanism in which the peptide is phosphorylated rapidly (154 s(-1)) and the product(s) is (are) released slowly (16 s(-1)). The replacement of Mg2+ with Mn2+ leads to a 13-fold reduction in this observed "burst" rate constant, suggesting that this transient is limited either by the phosphoryl transfer step or by a metal ion-dependent conformational change step. The influence of deuterium oxide on the pre-steady-state kinetics was monitored in the presence of both divalent metal ions, and no solvent isotope effect was measured on either "burst" phase. A large solvent isotope effect is observed on k(cat) in the presence of either metal ion, and a proton inventory analysis in the presence of Mg2+ indicates that two or more protons are transferred in the product release step. Finally, no pH dependence is observed on the "burst" rate constant using either Mg2+ or Mn2+ over the pH range of 6-9. The combined data do not support a mechanism involving a general-base catalyst whose pK(a) is greater than 5 or less than 10 if the "burst" phase is cleanly limited by the phosphoryl transfer step. If the "burst" phase is limited by a metal ion-dependent conformational change step, the measurement of the phosphoryl transfer step is obscured, and the participation of a base catalyst is indeterminate.

Adenosine Triphosphate↗
PubMed1997

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF) on megakaryopoiesis in normal subjects and patients with myelodysplasia and acute myeloid leukaemia.

Mpl ligand is a recently cloned haemopoietic growth factor that stimulates megakaryopoiesis in vitro and in vivo. We describe the in vitro effect of a truncated form of Mpl ligand, recombinant human megakaryocyte growth and development factor (rHuMGDF), on megakaryopoiesis in bone marrow from normal subjects and patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We used both semi-solid and suspension culture techniques to assess the effect of pegylated (PEG) rHuMGDF on megakaryocyte colony growth (CFU-Mk) and on the production of CD61+ cells in 7d suspension cultures. PEG rHuMGDF increased CFU-Mk growth and CD61+ cell production in a dose-dependent fashion in all normal marrows tested. Normal CFU-Mk growth was increased threefold with the addition of 10 ng/ml PEG rHuMGDF to cultures and CD61+ cells were increased 8-10-fold by the same dose. Although increased CFU-Mk growth was only seen in 1/10 AML and 6/16 MDS marrows, CD61+ cell numbers in suspension culture were increased in 9/13 AML and 12/15 MDS samples, responses ranged from very limited to normal magnitude. There was no correlation between platelet count and CFU-Mk number, CD61+ cell number or response to PEG rHuMGDF. We did not find any increased CFU-GM colony or cluster growth in response to PEG rHuMGDF and the CD61+ cells produced in suspension culture had features of megakaryocytic differentiation. These data suggest that PEG rHuMGDF can enhance megakaryocyte proliferation in some patients with MDS and AML, and may have a role in the treatment of thrombocytopenia in these patients.

Bone Marrow Cells↗
PubMed1997

Expression of AML1/MTG8 transcripts in clonogenic cells grown from bone marrow of patients in remission of acute myeloid leukaemia with t(8;21).

Patients in long-term remission of acute myeloid leukaemia (AML) M2 with t(8;21) after chemotherapy, with or without bone marrow transplantation, are known to retain residual cells which express AML1/MTG8 transcripts in bone marrow, detectable by RT-PCR. In order to determine whether these residual cells are clonogenic, we have grown remission bone marrow samples in standard semi-solid culture and picked individual CFU-GM and BFU-E colonies which were then analysed for the expression of AML1/MTG8 transcripts using a rapid specific RT-PCR technique. Nine patients were tested in remission, six between 1 and 83 months post chemotherapy, one 103 months post autologous bone marrow transplant and one 41 months post allogeneic bone marrow transplant. One of these patients also had quantitation of AML1/MTG8 transcripts on five occasions after recovery from each course of chemotherapy and at the end of treatment. There was a significant correlation between the percentage of positive colonies and the level of AML1/MTG8 transcripts. Between two and 80 CFU-GM and between two and 21 BFU-E colonies were analysed from each patient sample: 0-23% CFU-GM and 0-17% BFU-E colonies were found to express AML1/MTG8 transcripts suggesting that these residual cells are clonogenic in vitro and that the cell of origin is a multipotent myeloid progenitor.

Acute Disease↗
PubMed1997

The pharmacokinetics of bumetanide in the newborn infant.

This study characterizes the pharmacokinetics of bumetanide after an intravenous dose of 0.05 or 0.10 mg/kg to 14 neonates (weight range 820-4,000 g; gestational age 26-40 weeks) during the first week of life. Blood samples and urine were collected for up to 12 h after dosing. Estimated serum clearance was 0.2-1.0 ml/min.kg (range), volume of distribution was 0.22 l/kg (range 0.11-0.32 l/kg), and the harmonic mean half-life was 6-7 h (range of 4-19 h). Nonrenal clearance accounted for 58-97% of the serum clearance with the presence of certain oxidative metabolites of bumetanide in the urine. These findings suggest higher dosing requirements and prolonged intervals as compared to adults. Utilizing these pharmacokinetic data, pharmacodynamic and ototoxicity studies should be conducted to establish a safe and effective neonatal dose.

Bumetanide↗
PubMed1997

Hypoxemic events in spontaneously breathing premature infants: etiologic basis.

Studies of severe hypoxemic events, defined as an arterial oxygen saturation < 80% greater than 4 s in spontaneously breathing infants, have been limited. The purpose of our study was to examine the distribution of respiratory events that lead to a fall in oximetrically measured oxygen saturation by using breathing patterns, heart rate, and validated pulse oximetry analysis. A total of 161 hypoxemic events were detected in 18 of 30 premature infants studied. Using an inductive plethysmographic based monitor, a total of 460 h of cardiorespiratory monitor recordings were analyzed. Hypoxemic events were categorized as being the direct result of apnea (duration longer than 15 s) or pauses (duration 4-14 s) with either unchanged or lower end-expiratory lung volumes compared with the preevent breaths. The breaths in the preevent period were analyzed for volume, timing, and thoracoabdominal coordination indices. Forty of the 161 events (25%) were associated with apnea of which 80% (31/40) had a mixed/obstructive basis. Ninety-four of the 161 severe hypoxemic events (58%) were associated with pauses with unchanged end-expiratory lung volume. Twenty-two of the 161 events (14%) showed pauses with lower end-expiratory lung volume. There were 5/161 events (3%) with severe hypoxemia in which no pause was observed. Comparison of the preevent periods in each category showed significant differences for only percent tidal volume from initial calibration and arterial oxygen saturation. Sixty-two percent (100/161) of severe hypoxemic events were preceded by hypopneic values of percent tidal volume. Seventy-five percent (40/161) of these hypoxemic events and their etiology would have gone undetected using respiratory monitoring from impedance pneumograms and ECGs. The varied basis for these events underscores the importance of analyzing detailed respiratory wave forms along with movement-free signal of arterial oxygen saturation and ECG, to formulate appropriate intervention strategies.

Apnea↗
PubMed1996

Conformal irradiation of the prostate: estimating long-term rectal bleeding risk using dose-volume histograms.

PURPOSE: Dose-volume histograms (DVHs) may be very useful tools for estimating probability of normal tissue complications (NTCP), but there is not yet an agreed upon method for their analysis. This study introduces a statistical method of aggregating and analyzing primary data from DVHs and associated outcomes. It explores the dose-volume relationship for NTCP of the rectum, using long-term data on rectal wall bleeding following prostatic irradiation. METHODS AND MATERIALS: Previously published data were reviewed and updated on 41 patients with Stages T3 and T4 prostatic carcinoma treated with photons followed by perineal proton boost, including dose-volume histograms (DVHs) of each patient's anterior rectal wall and data on the occurrence of postirradiation rectal bleeding (minimum FU > 4 years). Logistic regression was used to test whether some individual combination of dose and volume irradiated might best separate the DVHs into categories of high or low risk for rectal bleeding. Further analysis explored whether a group of such dose-volume combinations might be superior in predicting complication risk. These results were compared with results of the "critical volume model," a mathematical model based on assumptions of underlying radiobiological interactions. RESULTS: Ten of the 128 tested dose-volume combinations proved to be "statistically significant combinations" (SSCs) distinguishing between bleeders (14 out of 41) and nonbleeders (27 out of 41), ranging contiguously between 60 CGE (Cobalt Gray Equivalent) to 70% of the anterior rectal wall and 75 CGE to 30%. Calculated odds ratios for each SSC were not significantly different across the individual SSCs; however, analysis combining SSCs allowed segregation of DVHs into three risk groups: low, moderate, and high. Estimates of probabilities of normal tissue complications (NTCPs) based on these risk groups correlated strongly with observed data (p = 0.003) and with biomathematical model-generated NTCPs. CONCLUSIONS: There is a dose-volume relationship for rectal mucosal bleeding in the region between 60 and 75 CGE; therefore, efforts to spare rectal wall volume using improved treatment planning and delivery techniques are important. Stratifying dose-volume histograms (DVHs) into risk groups, as done in this study, represents a useful means of analyzing empirical data as a function of hetereogeneous dose distributions. Modeling efforts may extend these results to more heterogeneous treatment techniques. Such analysis of DVH data may allow practicing clinicians to better assess the risk of various treatments, fields, or doses, when caring for an individual patient.

Dose-Response Relationship, Radiation↗
PubMed1996

Insight into tyrosine phosphorylation in v-Fps using proton inventory techniques.

The phosphoryl group transfer step in the kinase domain of v-Fps, a nonreceptor tyrosine protein kinase, was analyzed using proton inventory and viscosometric techniques. The latter studies show that two nine-residue peptide substrates for v-Fps, peptides I (EAEAYEAIE) and II (EAEIYEAIE), are in rapid equilibrium with the active site and bind with similar affinities (Ks = 2.2 and 1.7 mM for peptides I and II). While phosphoryl group transfer is the rate-limiting step in kcat for peptide I (5 s-1) at neutral pH, peptide II is converted to product by a kinetic mechanism in which phosphoryl group transfer (45 s-1) and product release (20 s-1) partially control this parameter. Significant solvent isotope effects on kcat (k0/kn approximately 1.6) are observed for the phosphorylation of both peptides in 95% D2O. Proton inventories on kcat for peptide I are linear, indicating that the phosphoryl group transfer step is associated with a single proton transfer. Conversely, proton inventories on kcat for peptide II are "bowed" up, consistent with a "virtual" transition state in which phosphoryl group transfer and product release steps partially control this parameter. The lack of solvent isotope effects on kcat/Kpeptide for both peptides can be explained by an equilibrium isotope effect on substrate binding that offsets the kinetic isotope effect for phosphoryl group transfer. In keeping with this proposal, the KI for the inhibitor peptide, EAEIFEAIE, is 11 +/- 0.80 and 6.5 +/- 0.82 mM in 0 and 60% D2O, respectively. Fitting of the proton inventory plots using modified forms of the Gross-Butler equation provide intrinsic isotope effects of 1.7 and 3.6 for peptides I and II. The combined data are consistent with a mechanism involving either an acid-base catalyst or a conformational change preceding the release of products that is accompanied by the disruption of a single hydrogen donor-acceptor pair.

Amino Acid Sequence↗
PubMed1996

Pre-steady-state kinetic analysis of cAMP-dependent protein kinase using rapid quench flow techniques.

The phosphorylation of a peptide substrate by the catalytic subunit of cAMP-dependent protein kinase was monitored over short time periods (2-1000 ms) using a rapid quench flow mixing device and a radioactive assay. The production of phosphokemptide [LRRAS(P)LG] as a function of time is characterized by a rapid "burst" phase (250 s-1) followed by a slower, linear phase (L/[E]t = 21 s-1) at 100 microM Kemptide. The amplitude of this "burst" phase varies linearly with the enzyme concentration and represents approximately 100% of the total enzyme concentration, indicating that the "burst" phase is not due to product inhibition. The observed rate constants for the "burst" and linear phases and the "burst" amplitude vary hyperbolically with the substrate concentration. From these dependencies, a maximum "burst" rate constant of 500 +/- 60 s-1 and a Km and Kd for Kemptide of 4.9 +/- 1.4 and 200 +/- 60 microM were determined. The kcat and Km data extracted from the linear portion of the rapid quench flow transients are indistinguishable from those obtained by standard steady-state kinetic analyses using low catalytic subunit concentrations and a spectrophotometric, coupled enzyme assay. Both rate constants for the "burst" and linear phases decreased in the presence of Mn2+. The data imply that the phosphorylation of Kemptide by the catalytic subunit occurs by a mechanism in which the substrate is loosely bound, is rapidly phosphorylated at the active site, and is released at a steady-state rate that is likely controlled by the dissociation rate constant for ADP. The combined pre-steady-state kinetic data establish a comprehensive, kinetic mechanism that predicts all the steady-state kinetic and viscosometric data. This study represents the first chemical observation and characterization of phosphoryl transfer at the active site of a protein kinase and will be useful for further structure-function studies on this and other protein kinases.

Amino Acid Sequence↗
PubMed1996

Rate-determining steps for tyrosine phosphorylation by the kinase domain of v-fps.

The rate-determining steps in the phosphorylation of four tyrosine-containing peptides by the kinase domain of the nonreceptor tyrosine protein kinase v-fps were measured using viscosometric methods. The peptides were phosphorylated by a fusion protein of glutathione-S-transferase and the kinase domain of v-fps (GST-kin) and the initial velocities were determined by a coupled enzyme assay. Peptides I (EEEIYEEIE), II (EAEIYEAIE), and III (DADIYDAID) were phosphorylated by GST-kin with similar kinetic constants. The viscosogens, glycerol and sucrose, were found to have intermediate effects on kcat and no effect on kcat/Kpeptide for the phosphorylation of these three peptides. The data are interpreted according to the Stokes-Einstein equation and a simple three-step mechanism involving substrate binding, phosphoryl group transfer, and net product release. Two competitive inhibitors (EAEIFEAIE and DADIFDAID) exhibited K1 values that are 6-10-fold higher than the Kpeptide values for their analogous peptide substrates. The data imply that peptides I-III are in rapid equilibrium with the enzyme and that kcat is partially limited by both phosphoryl group transfer (40-100 s-1) and product release (17-22 s-1). GST-kin phosphorylates peptide IV (R5AENLEYamide) with a low Km (100 microM) and a kcat that is 40-fold lower than that for peptide I. No effect of solvent viscosity was observed for the phosphorylation of this peptide on either kcat or kcat/Kpeptide. This suggests that highly viscous solutions do not perturb structure and that the rate-determining step for this poor substrate is phosphoryl group transfer. The data indicate that the kinase domain of v-fps phosphorylates its best substrate with a chemical rate constant that is at least 5-fold lower than that for the serine-specific cAMP-dependent protein kinase and its best substrate LRRASLG (Adams & Taylor, 1992). Interestingly, both enzymes exhibit a similar affinity for their substrates and both enzymes release their products at a similar rate. This implies that the differences in catalytic efficiency between serine- and tyrosine-specific protein kinases lie exclusively in the rate constants for phosphoryl group transfer and not in substrate absorption or product desorption.

Amino Acid Sequence↗
PubMed1996

Genital findings in adolescent girls referred for suspected sexual abuse.

BACKGROUND: Sexual abuse is a common problem affecting adolescent girls, but the frequency of medical findings in this population has not been specifically described. OBJECTIVE: To describe the frequency of specific genital findings in a group of pubertal girls who had experienced probable or definite sexual abuse. DESIGN: Patient series, medical chart and photograph review. SETTING: Specialty referral clinic for abused children. PATIENTS AND SELECTION: Referred sample of female patients, examined between January 1, 1987, and June 30, 1994, with Tanner genital stages 3, 4, or 5, who reported a history of penile-vaginal penetration, had colposcopic photographs taken, and were determined, by means of a previously described classification system, to have experienced probable or definite abuse. INTERVENTIONS: None. MAIN RESULTS: The study included 204 girls, aged 9 to 17 years (mean, 13 years); race or ethnicity was Mexican American in 57%, white in 34%, and other in 9%. Abnormal genital findings were documented in 32% of patients overall but were more common when the girls had reported bleeding at the time of the assault (50% vs 26%; P = .004, chi 2 analysis), or when the examination occurred within 72 hours of the last episode of abuse (69% vs 26%; P < .001, chi 2 analysis). Transections of the hymen were unusual (8%), but notches in the hymen were more common (25%). CONCLUSIONS: Normal or nonspecific results of genital examinations are commonly found in adolescents who have been sexually abused, unless the abuse was very recent. Further studies are needed to document the healing of genital injuries in victims of acute assault and the frequency of hymenal findings in nonabused, non-sexually active adolescents.

Adolescent↗
PubMed1996

Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit.

The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.

Animals↗
PubMed1996

Therapeutic play and bone marrow transplantation.

Constructive, therapeutic play is an essential part of the care of children with long-term hospitalizations. The O'Connor theoretical framework supports the importance of play in ensuring the emotional, developmental, and physical health of children. The negative effects of long-term hospitalization are particularly evident for children who have undergone bone marrow transplants and must be kept in germ-free environment and isolation for extended periods of time. This article describes a successful play therapy program in a Bone Marrow Transplant Unit, using a play cabinet designed to provide readily available, sterilized toys that are appropriate for each of four age groups. Two cases are presented that show the efficacy of the use of the play cabinet in play therapy programs.

Bone Marrow Transplantation↗
PubMed1996

The effect of the chemokine rhMIP-1 alpha, and a non-aggregating variant BB-10010, on blast cells from patients with acute myeloid leukaemia.

The effects of recombinant macrophage inflammatory protein 1 alpha (rhMIP-1 alpha) on the proliferation of leukaemic blast cells from patients with acute myeloid leukaemia was assessed. Using the previously described [3H]thymidine incorporation index assay, the response of autonomous and growth factor responsive AML blast cells to the chemokine rhMIP-1 alpha was measured. In the case of autonomous proliferators, rhMIP-1 alpha had no inhibitory effect on [3H]thymidine incorporation and in 4/6 cases [3H]-thymidine incorporation was stimulated by rhMIP-1 alpha. In the presence of stem cell factor (SCF), a majority (8/9) of the samples which responded to this growth factor were inhibited when rhMIP-1 alpha was included in the assay medium. Similar results were obtained with GM-CSF-responsive samples; however, when these two cytokines were combined, only 3/14 were significantly inhibited. In the presence of human placental conditioned medium (HPCM), rhMIP-1 alpha significantly inhibited [3H]thymidine incorporation in only 2/10 of HPCM-responsive samples. In methylcellulose assays rhMIP-1 alpha had no consistent effect on colony/cluster formation in the presence of either GM-CSF + SCF or HPCM. Similar results were obtained with BB-10010, a mutant of rhMIP-1 alpha which has defined aggregation properties in solution. These data suggest that autonomously proliferating AML cells, and also some AML samples which require cytokines to proliferate, are non-responsive to the growth inhibitors rhMIP-1 alpha and BB-10010 in the presence of multiple growth factors.

Acute Disease↗