Biomedical subjects
Publications and source records attributed to R Lim.
This randomized study using a pneumatic compression device found no significant difference in the femoral complication rate between 4 and 6 hours of bed rest after Judkin's coronary arteriography. The positive implications for the organization of an efficient service in busy tertiary centers include reduced patient discomfort, earlier ambulation and discharge, efficient staff deployment, and enhanced throughput.
We observed previously that glia maturation factor (GMF), a 17-kDa brain protein, is rapidly phosphorylated in astrocytes following stimulation by phorbol ester, and that protein kinase A (PKA)-phosphorylated GMF is a potent inhibitor of extracellular signal-regulated kinase (ERK) and enhancer of p38; both are subfamilies of mitogen-activated protein (MAP) kinase, suggesting GMF as a bifunctional regulator of the MAP kinase cascades. In the current report, we present evidence that PKA-phosphorylated GMF also promotes (11-fold) the catalytic activity of PKA itself, resulting in a positive feedback loop. Furthermore, GMF phosphorylated by protein kinase C (PKC), but not by casein kinase II or p90 ribosomal S6 kinase, also activates PKA (7-fold). It appears that the mutual augmentation of GMF and PKA, and the stimulating effect of PKC, both serve to maximize the influence of PKA on the regulation of MAP kinase cascades by GMF. Using synthetic peptide fragments containing putative phosphorylation sites of GMF, we demonstrate that PKA is capable of phosphorylating threonine 26 and serine 82, whereas PKC, p90 ribosomal S6 kinase, and casein kinase II, can phosphorylate serine 71, threonine 26, and serine 52, respectively. The generation of various phospho-isoforms of GMF may explain its modulation of signal transduction at multiple locations.
OBJECTIVES: To evaluate the palatability of antibiotics effective against beta-lactamase-producing bacteria in children and to compare the results obtained with those obtained in adults. DESIGN: A taste test of 4 antibiotic suspensions: a combination of amoxicillin and clavulanic acid (banana), azithromycin (cherry), clarithromycin (wild fruit), and a combination of erythromycin and sulfisoxazole (strawberry-banana). SETTING: Outpatient setting. SUBJECTS: A volunteer sample of 50 healthy children (mean +/- SD age, 6.3 +/- 1.3 years) and 20 adults. MAIN OUTCOME MEASURES: After each antibiotic test dose, subjects rated its taste on a 10-cm visual analog scale incorporating a facial hedonic scale. RESULTS: The mean +/- SD taste scores of the antibiotics as rated by the children were as follows: amoxicillin-clavulanic acid, 5.7 +/- 3.6 cm; azithromycin, 6.8 +/- 3.2 cm; clarithromycin, 3.7 +/- 3.6 cm; and erythromycin-sulfisoxazole, 4.9 +/- 3.5 cm. The mean +/- SD taste score for erythromycin-sulfisoxazole (ie, 2.7 +/- 2.3) assigned by the adults was significantly different than that given by the children (P = .01) with no difference noted for the other 3 drugs. Children and adults both selected azithromycin most often as best tasting. There was a significant difference in the proportions selecting each antibiotic as worst tasting, with the children tending to dislike clarithromycin and the adults tending to dislike erythromycin-sulfisoxazole (P = .03). CONCLUSIONS: The taste of azithromycin was rated most highly by both children and adults, who also selected this antibiotic most often as best tasting. Differences in taste-testing results between children and adults suggest that evaluation of the palatability of medications intended for use in pediatrics should be conducted in children.
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We previously demonstrated that glia maturation factor (GMF), a 17-kDa brain protein, can be phosphorylated in test tube by several protein kinases, and that endogenous GMF is rapidly phosphorylated upon stimulation of astrocytes by phorbol 12-myristate 13-acetate. We further observed that protein kinase A (PKA)-phosphorylated GMF is a potent inhibitor (IC50 = 3 nM) of the ERK1/ERK2 (p44/p42) subfamily of mitogen-activated protein (MAP) kinase. We now report that, by contrast, PKA-phosphorylated GMF strongly enhances the activity of a related but distinct subfamily of MAP kinase, the p38 MAP kinase, showing an increase of 60-fold over baseline and an EC50 of 7 nM. Non-phosphorylated GMF or GMF phosphorylated by other kinases exhibits only minimal effect. The intracellular interaction of PKA, GMF, and p38 is supported by the phosphorylation of GMF upon cellular stimulation by forskolin (blocked by PKA inhibitor) and by the co-immunoprecipitation of p38 with GMF from cell lysates. Withdrawal of nerve growth factor from PC12 leads to increased GMF phosphorylation with a time course similar to that reported for p38 activation. The results correlate well with a previous report that ERK and p38 carry out opposing functions and implicate GMF as a regulator of major cellular events.
We report that recombinant glia maturation factor (GMF), a 17-kDa brain protein, inhibits the activity of mitogen-activated protein (MAP) kinase in the test tube assay, in particular the ERK1/ERK2 isoforms. A preliminary phosphorylation of GMF by protein kinase A (PKA) dramatically increases its inhibitory effect by over 600-fold (Ki approximately 3 nM), making it the most potent MAP kinase inhibitor ever reported. Immunoprecipitation of GMF from cell extracts using its specific antibody coprecipitates ERK (and vice versa), suggesting the association of the two proteins in the cell. The inhibitory effect of PKA-phosphorylated GMF is specific, as it does not suppress the activity of cdc2 kinase, another proline-directed kinase. Nor does it inhibit MAP kinase kinase (MEK) and MAP kinase-activated protein (MAPKAP) kinase-2, the two enzymes immediately upstream and downstream, respectively, of ERK. Of the other three enzymes that can phosphorylate GMF, only p90 ribosomal S6 kinase (RSK) enhances the inhibitory function of GMF on ERK; protein kinase C (PKC) and casein kinase II (CKII) are without effect. The inhibition of ERK by PKA-phosphorylated GMF suggests that GMF could be one of the mediators of the suppressive effect of the PKA pathway on the MAP kinase pathway. On the other hand, that RSK-phosphorylated GMF also inhibits ERK implies a negative feedback loop in the regulation of MAP kinase activity.
An approach to restriction-site mapping and contig building that uses fragment-size data from multiple complete digests of a set of clones that oversample a genomic region is presented. Maps containing both fragment-length data and clone-end data are maintained for each restriction enzyme. Synchronization between the maps for the different enzymes is achieved by requiring the clone-end maps for all enzymes to be compatible. Basic concepts that underlie multiple-complete-digest mapping--including the match/merge approach to map incorporation, extension vs assimilation, ambiguity, and clone-end compatibility--are presented. An initial application of multiple-complete-digest mapping to real data on a set of cosmid clones suggests that this mapping method has exceptional power to produce accurate maps that are well suited to the needs of large-scale DNA-sequencing projects.
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Adult rats with bilateral thermal lesions of the caudate nuclei (CN) show severe learning and memory deficits. The present study was designed to test the effects of an astroglial stimulating growth factor in this behavioral model. Immediately after receiving lesions of the CN, experimental subjects received an injection of one of three doses of glial maturation factor-beta (GMF-beta) directly in the lesion site. All subjects were then tested for twenty days on an active avoidance spatial alternation task. The behavioral recovery of the three groups of experimental animals was compared to that of animals having received the same brain damage and administration of a control substance (lesion controls), and to that of animals receiving a sham operation and no treatment (shams). The beneficial effects of administration were evident in the group of experimental animals receiving the lowest dose of GMF-beta. The performance of animals in this group was indistinguishable from that of the shams, and was significantly better than that of the lesion controls. The results suggest a behavioral role of GMF-beta which, in an in vitro system, is known to be a growth regulator of astroglial cells.
OBJECTIVES: The concept of "cardioprotection" based on ejection fraction was tested to see whether patients with coronary artery disease in whom medical treatment fails to be cardioprotective can be distinguished from those in whom it is safe to continue such treatment. BACKGROUND: Ejection fraction is of fundamental prognostic importance. Its modification by anti-ischemic medication may allow assessment of cardioprotection from adverse outcome. METHODS: Exercise ejection fraction and the change in ejection fraction from rest to exercise were measured by radionuclide ventriculography with and without background medication in 102 mildly symptomatic patients with coronary artery disease suitable for revascularization but initially treated medically. RESULTS: Over 20 months, 23 patients experienced an adverse event. With medication, exercise ejection fraction increased in patients with and without events. By contrast, the ejection fraction response to exercise improved significantly in the event-free group only; the group with events had a persistent decrease in ejection fraction. By Cox analysis, the ejection fraction response to exercise performed with medication made the most significant independent contribution to event-free survival. Comparison of areas under receiver operating characteristic curves suggested that this index is the most useful clinical measure of cardioprotection. CONCLUSIONS: An exercise-induced decrease in ejection fraction despite anti-ischemic medication implies failure of cardioprotection and a greater short-term risk of adverse outcome and crossover to revascularization in patients initially treated medically. Conversely, a preserved left ventricular performance confers a satisfactory prognosis while continuing with that treatment. Thus, the effect of medication on the ejection fraction response to exercise--a reasonable estimate of its cardioprotective efficacy--may influence the choice of continuing with such treatment or performing early revascularization.
We report that recombinant glia maturation factor (GMF), a 17-kD brain protein, can be phosphorylated in vitro at the serine residue by protein kinase C (PKC), protein kinase A (PKA), and casein kinase II (CKII), and at the threonine residue by p90 ribosomal S6 kinase (RSK). Endogenous GMF in astrocytes is phosphorylated at the serine (major) and threonine (minor) residues within 15 min after stimulation by phorbol 12-myristate 13-acetate (PMA). Phosphorylation gradually subsides over the next 24 h. The increased phosphorylation is not blocked by the protein synthesis inhibitor cycloheximide and is not accompanied by a rise in the mRNA for GMF and is therefore strictly a posttranslational regulatory phenomenon. The rapid and transient phosphorylation of GMF upon cellular activation suggests an intracellular role, possibly with involvement in signal transduction.
The paradigm of sequence-tagged site (STS)-content mapping involves the systematic assignment of STSs to individual cloned DNA segments. To date, yeast artificial chromosomes (YACs) represent the most commonly employed cloning system for constructing STS maps of large genomic intervals, such as whole human chromosomes. For developing a complete YAC-based STS-content map of human chromosome 7, we wished to utilize a limited set of YAC clones that were highly enriched for chromosome 7 DNA. Toward that end, we have assembled a human chromosome 7 YAC resource that consists of three major components: (1) a newly constructed library derived from a human-hamster hybrid cell line containing chromosome 7 as its only human DNA; (2) a chromosome 7-enriched sublibrary derived from the CEPH mega-YAC collection by Alu-polymerase chain reaction (Alu-PCR)-based hybridization; and (3) a set of YACs isolated from several total genomic libraries by screening for > 125 chromosome 7 STSs. In particular, the hybrid cell line-derived YACs, which comprise the majority of the clones in the resource, have a relatively low chimera frequency (10-20%) based on mapping isolated insert ends to panels of human-hamster hybrid cell lines and analyzing individual clones by fluorescence in situ hybridization. An efficient strategy for polymerase chain reaction (PCR)-based screening of this YAC resource, which totals 4190 clones, has been developed and utilized to identify corresponding YACs for > 600 STSs. The results of this initial screening effort indicate that the human chromosome 7 YAC resource provides an average of 6.9 positive clones per STS, a level of redundancy that should support the assembly of large YAC contigs and the construction of a high-resolution STS-content map of the chromosome.
Neurons and glia are capable of both secreting and responding to a large variety of growth factors. However, information on multiple expression of growth factors and their receptors was usually obtained from uncorrelated observations, using cells from various animals of origin, developmental stages, growth phases, culture ages and culture conditions. Because of its specificity and extreme sensitivity, reverse transcription-polymerase chain reaction (RT-PCR) is uniquely suitable to study a large panel of growth factors and their receptors from a limited cell sample, free of these intervening variables. In this paper we evaluate the expression of mRNA of a total of 35 growth factor-related proteins by conducting RT-PCR on three neuronal cell lines: the PC12 rat pheochromocytoma line, the MAH rat sympathoadrenal progenitor line, and the N18 mouse neuroblastoma line. Three types of results are presented. The first confirms the existing knowledge such as the presence of Trk-A (NFG receptor) in PC12. The second consists of new information that expands and extends earlier observations, such as the presence of CNTF receptor complex in PC12, which explains our previous report that CNTF enhances the biological effects of NGF on these cells. The third consists of novel information that leads the way to further experimentation by the more conventional methods. These include the strong expression of Trk-B by MAH, predicting the biological responsiveness of MAH to BDNF and NT-4, and the expression of CNTF receptor in N18. Our results also suggest that CNTF is an autocrine factor for PC12 and MAH, since both lines express the growth factor as well as the receptor. Thus, RT-PCR is a valuable tool in growth factor research that can be used in complement to, and interactively with, other approaches such as bioassay, receptor binding, and immunochemical determination. It will be particularly useful for screening a large number of growth factors in minute areas of the brain in patients suffering from neurodegenerative diseases such as Parkinson's and Alzheimer's.
1. Although glial cells in culture are known to secrete growth factors and are also known to be responsive to some of them, detailed comparisons are difficult because the bulk of information was based on various animals of origin, developmental stages, growth properties, culture age, and culture conditions. 2. To present a unified picture of the growth factors and their receptors found in glial cells, we surveyed the expression of messenger RNAs of a panel of growth factors and receptors, using reverse transcription-polymerase chain reaction (RT-PCR), in three common glial cell types: rat astrocytes in primary culture, rat glioma line C6, and human glioma line A172. 3. We observed that normal and neoplastic glial cells in culture express multiple growth factors and also possess most of the receptors to the factors, suggesting multiple autocrine functions. In addition, glia produce growth factors known to be capable of acting on neurons, implicating paracrine function involving glia-neuron interaction. Glial cells also produce growth factors and receptors that are capable of communicating with hematopoietic cells, suggesting neuroimmunologic interaction. What is most interesting is that glial cells express receptors for growth factors previously thought to be acting on neurons only. 4. The current study demonstrates the feasibility of screening from a small sample a large number of growth factors and receptors. The method portends future clinical application to biopsy or necropsy samples from brain tumors or pathologic brains suffering from degenerative diseases such as Alzheimer's or Parkinson's disease.
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