Transgenic mice for the preparation of hygromycin-resistant primary embryonic fibroblast feeder layers for embryonic stem cell selections.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
K A Johnson
Publications and source records attributed to K A Johnson.
Pathway of processive ATP hydrolysis by kinesin.
Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin-ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin.
Adenosine Diphosphate↗
Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors.
The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and CI-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady-state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (Kd, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very tightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS.
Antiviral Agents↗
Tibial intercalary allograft incorporation: comparison of fixation with locked intramedullary nail and dynamic compression plate.
The healing and incorporation of intercalary cortical bone allografts stabilized with locked intramedullary nails was compared with that of allografts fixed with compression plates in a sheep model. Both techniques achieved healing of the allograft to host tibia, and there was no measurable difference in the area of callus or the quality of union histologically. Incorporation of the allograft as measured by fluorescent labeling was not different in the two groups.
Animals↗
Oculoauriculovertebral spectrum: new manifestations.
We present a case of oculoauriculovertebral spectrum (hemifacial microsomia, Goldenhar's syndrome) found shortly after birth with right iliac hypoplasia, absent right pubic and ischial ossification centres and posterior urethral valves, which have not previously been described in this spectrum.
Abnormalities, Multiple↗
Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates.
Explore the source record for details and available documents.
Actomyosin↗
One framework, two systems: flexible abductive methods in the problem-space paradigm applied to antibody identification and biopsy interpretation.
Our goal is to build flexible knowledge-based systems which can use a variety of problem-solving methods and additional task knowledge, without altering the method or task representation. For this purpose, we use a problem-space architecture which allows opportunistic adaptation of problem-solving methods based on the particular goal, situation and knowledge available. Within this framework, we have developed an opportunistic problem-solving method for flexible abductive problem solving. The basic method was specified in terms of potential subgoals and preferences regarding the order of subgoals. This technique avoids specification of any unnecessary procedural commitments, making the resulting method very general and robust. We then developed two systems using this basic abductive method with additional domain-dependent search-control and task knowledge. The additional knowledge alters/overrides some of the minimal knowledge provided by the basic method. Behavior of these systems can change quite dramatically depending on the added increments of knowledge. Knowledge available at runtime shapes the method and hence the behavior. Moreover, even in the absence of strong domain knowledge, due to the wide coverage of the basic abductive method and the general architecture it is based on, the systems are always able to perform some problem solving--they are not brittle. The specific systems implemented are RedSoar and LiverSoar in the domain of red blood cell antibody identification and liver biopsy interpretation, respectively. We discuss the designs, implementations and evaluations of these systems, emphasizing the role the flexible abductive problem-solving method plays.
Antibodies↗
Antidepressant drug-induced hypothalamic cooling in Syrian hamsters.
Antidepressant drugs have been reported to alter the circadian pattern of body temperature, but specific effects on the amplitude or on average body temperature are not consistent, and there have been no specific studies to examine chronic drug effects on brain temperature. To address these issues, hypothalamic temperature (Thy) was monitored telemetrically in hamsters treated with three antidepressant drugs: the monoamine oxidase inhibitor (MAOI), clorgyline; the 5HT reuptake inhibitor, fluoxetine; and the alkali metal, lithium. For comparison, hamsters were also treated with two neuroleptic drugs, chlorpromazine and haloperidol. Each of the three antidepressant drugs, but neither of the neuroleptic drugs, produced a chronic decrease in diurnal (rest-phase) hypothalamic temperature. The Thy-decreasing effect of clorgyline was not prevented by pinealectomy, and Thy decreased more than peritoneal temperature (Tp), thus reducing the temperature difference between the hypothalamus and the peritoneal cavity. Less general effects of the antidepressants were also observed. Clorgyline and fluoxetine, but not lithium, delayed the 24-hour rhythm of Thy. Clorgyline and lithium, but not fluoxetine decreased the average 24-hour Thy. The neuroleptics chlorpromazine and haloperidol decreased the amplitude of the 24-hour Thy rhythm. The fact that chronic antidepressant drugs, but not neuroleptic drugs, decrease Thy is consistent with their different neurotransmitter effects and clinical applications, and raises the possibility that their antidepressant property might be related to their capacity to decrease Thy during sleep.
Animals↗
Double dissociation of memory capacities after bilateral occipital-lobe or medial temporal-lobe lesions.
Memory for recently encountered information can be reflected in conscious recall and recognition of that material, or in facilitated reprocessing of that material, an effect known as repetition priming. Repetition priming may be perceptual (form-based) or conceptual (meaning-based). A patient with bilateral occipital-lobe lesions (L.H.) and a patient with bilateral medial-temporal lobe lesions (H.M.) showed a double dissociation between visuoperceptual priming (impaired in L.H. and intact in H.M.) and visual recognition memory (intact in L.H. and impaired in H.M.). L.H. showed intact conceptual priming for visually presented words; his pattern of impaired visuoperceptual priming and intact conceptual priming is the reverse dissociation to that observed in prior studies of patients with Alzheimer's disease, in whom occipital cortices are relatively spared. These double dissociations suggest that a memory system localized to the occipital lobe mediates visuoperceptual priming effects, and that this system is independent of neural circuits mediating conceptual priming effects, and independent of the limbic-diencephalic system supporting conscious recognition of recently encountered information.
Adult↗
Comparison of hemilaminectomy and dorsal laminectomy for thoracolumbar intervertebral disc extrusion in dachshunds.
Neurological improvement after decompressive surgery, without routine therapeutic or prophylactic disc fenestration, was evaluated retrospectively in a consecutive series of 93 dachshunds with thoracolumbar disc extrusion. In 24 per cent of procedures, deep pain sensation was not elicited in at least one hind paw before surgery. Median neurological status one to seven days after surgery, at the time of discharge, was significantly improved after hemilaminectomy compared with dorsal laminectomy (P < 0.05). After hemilaminectomy, deep pain sensation was not elicited in one or both hind paws of 8 per cent of dogs in which pain sensation was elicited before surgery, compared with 21 per cent after dorsal laminectomy (P > 0.05). Improvement in neurological grade at follow-up examination two to 12 weeks after hemilaminectomy was not significantly different compared with dorsal laminectomy (P > 0.05). Of dogs which were unable to walk before surgery, 83 per cent regained the ability to walk after hemilaminectomy, compared with 74 per cent after dorsal laminectomy (P > 0.05). In both groups, 50 per cent of dogs in which deep pain sensation was abnormal before surgery eventually regained the ability to walk after surgery (P = 1). One to two years after the first operation, a second laminectomy was performed in only 5 per cent of dogs because of extrusion of a different intervertebral disc which had not been fenestrated.
Animals↗
SPECT perfusion imaging in the diagnosis of dementia.
Single-photon emission computed tomography (SPECT) imaging has provided the practicing clinician with a method of studying brain function in patients with dementia. A large and growing number of papers report the experiences of a number of laboratories in the use of this technique in the evaluation of demented patients. Studies from several laboratories comparing patients with Alzheimer's disease to control subjects report sensitivity and specificity of SPECT perfusion imaging to be in the 80% vicinity. In addition, a number of studies suggest that the dementias that show the greatest similarities in perfusion patterns to Alzheimer's disease are multi-infarct dementia and dementia associated with Parkinson's disease. Although considerable data exists to guide the physician, a rigorous scientific approach to studying patients in a prospective, unselected clinical sample, with autopsy confirmation of the diagnosis, is needed to define clearly the utility of the technique in diagnosing dementias.
Alzheimer Disease↗
The perceptual representation of voice gender.
The perceptual representation of voice gender was examined with two experimental paradigms: identification/discrimination and selective adaptation. The results from the identification and discrimination of a synthetic male-female voice continuum indicated that voice gender perception was not categorical. In addition, results from selective adaptation experiments with natural and synthetic voice stimuli indicated that the perceptual representation of voice adapted is an auditory-based representation. Overall, these findings suggest that the perceptual representation of voice gender is auditory based and is qualitatively different from the representation of phonetic information.
Female↗
Identification of a molecular chaperone in the eukaryotic flagellum and its localization to the site of microtubule assembly.
Monoclonal antibodies that recognize HSP70 family members from evolutionarily divergent organisms were used to identify both constitutively-expressed and stress-inducible HSP70 proteins in the green alga Chlamydomonas. These monoclonal antibodies also cross-reacted with a 70 kDa flagellar protein that comigrated with the constitutively-expressed HSP70 isoform(s) present in the cell body; this is the first identification of a molecular chaperone within the eukaryotic flagellum. Fractionation experiments demonstrated that much of the flagellar HSP70 was bound to the '9+2' microtubule axoneme. Incubation of isolated axonemes in ATP, but not AMP or AMP-PNP, caused significant release of the previously bound HSP70 as is characteristic of complexed HSP70s. Immunofluorescent localization in whole flagella showed that flagellar HSP70 was concentrated at the distal ends of flagella, sites of axonemal assembly in vivo. Extraction of axonemes under ionic conditions known to cause the release of capping structures that link the distal ends of the axonemal microtubules to the flagellar membrane also caused the release of axonemal-bound HSP70. Taken together, these results suggest a model in which an HSP70 chaperone may assist in targeting tubulin and other unassembled axonemal components to the flagellar tip where the chaperone may also participate in the assembly of the '9+2' flagellar axoneme.
Adenosine Triphosphate↗
Methane emissions from cattle.
Increasing atmospheric concentrations of methane have led scientists to examine its sources of origin. Ruminant livestock can produce 250 to 500 L of methane per day. This level of production results in estimates of the contribution by cattle to global warming that may occur in the next 50 to 100 yr to be a little less than 2%. Many factors influence methane emissions from cattle and include the following: level of feed intake, type of carbohydrate in the diet, feed processing, addition of lipids or ionophores to the diet, and alterations in the ruminal microflora. Manipulation of these factors can reduce methane emissions from cattle. Many techniques exist to quantify methane emissions from individual or groups of animals. Enclosure techniques are precise but require trained animals and may limit animal movement. Isotopic and nonisotopic tracer techniques may also be used effectively. Prediction equations based on fermentation balance or feed characteristics have been used to estimate methane production. These equations are useful, but the assumptions and conditions that must be met for each equation limit their ability to accurately predict methane production. Methane production from groups of animals can be measured by mass balance, micrometeorological, or tracer methods. These techniques can measure methane emissions from animals in either indoor or outdoor enclosures. Use of these techniques and knowledge of the factors that impact methane production can result in the development of mitigation strategies to reduce methane losses by cattle. Implementation of these strategies should result in enhanced animal productivity and decreased contributions by cattle to the atmospheric methane budget.
Animal Feed↗
Pathway of the microtubule-kinesin ATPase.
We have established pathway of the kinesin ATPase by direct measurement of each step in the pathway. Kinesin binds to microtubules with an 8-nm repeat and a stoichiometry of one kinesin monomer unit per tubulin dimer. Thus, the dimeric kinesin binds with both heads attached to the microtubule and on adjacent tubulin subunits. In the steady state, kinesin has a low ATPase activity that is limited by the rate of ADP release (< 0.01 s-1) in the absence of microtubules and is activated 2000-fold by the addition of microtubules to achieve a maximum rate of approximately 20 s-1. Transient-state kinetic analysis has provided direct measurement of individual steps of the reaction to define the pathway of the microtubule-kinesin ATPase. These studies establish that the rate-limiting step in the ATPase pathway is the release of the kinesin-product complex (K.ADP.P) from the microtubule following ATP hydrolysis. After phosphate release, the rebinding of kinesin-ADP to the microtubule is fast, accounting for the high activation of the ATPase at low microtubule concentration. This ATPase cycle explains the phenomenological differences between myosin and kinesin observed in motility assays. Kinesin remains associated with a microtubule through multiple rounds of hydrolysis, because it spends only a small fraction of its duty cycle in the dissociated state. The discussion of this paper will focus on the new data, their interpretation, and significance for mechanisms of force production. The ATPase coupling mechanism will be compared with dynein and myosin.
Adenosine Triphosphatases↗
Mutants affecting nucleotide recognition by T7 DNA polymerase.
Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in misincorporation assays. Both E480D(exo-) and Y530F(exo-) showed saturation kinetics with the wrong nucleotide, with binding constants of 1-3 mM compared to the estimated binding affinity of 6-8 mM with wild-type(exo-). Accordingly, both mutants showed slightly lower selectivity against misincorporation. Taken together, these results indicate that E480 and Y530 each contribute to ground state nucleotide binding and suggest that the E480 may serve to specifically stabilize the incoming dTTP of A:T base pairs to compensate for the fewer hydrogen bonds compared to G:C base pairs.
Base Sequence↗
The potential for hormonal prevention trials.
Breast and prostate cancer are significant causes of morbidity and mortality and are very similar in etiology, epidemiology, and modalities of treatment. Investigational strategies in the prevention of these malignancies also have strong parallels. The National Cancer Institute is sponsoring several large scale clinical trials involving hormonal manipulation and cancer prevention. In the Breast Cancer Prevention Trial, 16,000 women at high risk for breast cancer are being randomized to receive the antiestrogen agent tamoxifen or placebo for 5 years in an effort to determine if breast cancer development can be inhibited. In a similar trial, the Prostate Cancer Prevention Trial, 18,000 men older than 55 years of age will be randomized to receive finasteride, a 5-alpha-reductase inhibitor, or placebo to determine if inhibition of dihydrotestosterone synthesis in the prostate over a prolonged period will lead to a decreased incidence of prostate cancer. Both clinical trials offer the possibility of demonstrating that a hormonal intervention can decrease an individual's risk of developing breast or prostate cancer. They also have the potential of providing critical information about cancer risk, etiology, screening, and genetics, as well as quantifying the risks and benefits of specific preventive interventions.
5-alpha Reductase Inhibitors↗