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Biomedical subjects

G Walter

Publications and source records attributed to G Walter.

At least 73 records · Page 4Linked to original sources
PubMed1999

Practitioner review: electroconvulsive therapy in adolescents.

There is increasing interest regarding electroconvulsive therapy (ECT) in young persons but limited information about its optimal use. This paper reviews the indications, effectiveness, and side effects of the treatment in the teenage population and their implications for everyday clinical practice. There is particular reference to factors influencing the clinician to recommend or advise against convulsive therapy, steps in the assessment of patients, and consent issues. Practical aspects of ECT administration are also examined, including treatment locale, anaesthesia, electrode position, stimulus characteristics, EEG monitoring, and use of psychotropics during and after the ECT course.

Adolescent↗
PubMed1999

Selective enrichment and high-throughput screening of phage surface-displayed cDNA libraries from complex allergenic systems.

Phage surface display technology, first described in 1985, enables the construction of large combinatorial peptide and antibody libraries. The basic concept of linking the phenotype, expressed as gene product displayed on the phage surface, to its genetic information integrated into the phage genome, allows the survey of large libraries for the presence of specific clones using the discriminative power of affinity purification. The selection procedure involves the enrichment of phage by binding to an immobilized target molecule. As a consequence of the physical linkage between genotype and phenotype, sequencing the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of a displayed gene product. Phage surface display technology has revolutionized our ability to select agonist and antagonist peptides, antibodies with desired specificities, and DNA-binding molecules. We have extended phage surface display to access cDNA libraries with this powerful screening technology based on affinity selection of desired clones. Here we discuss construction of cDNA libraries displayed on the phage surface, selective enrichment of clones and robotics-based high-throughput screening of enriched libraries.

Allergens↗
PubMed1998

Protein phosphatase 2A is required for the initiation of chromosomal DNA replication.

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.

Animals↗
PubMed1998

A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.

We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins.

Antibodies, Monoclonal↗
PubMed1998

Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis.

Although the available evidence suggests that whereas the caspase family plays a major role in apoptosis, they are not the sole stimulators of death. A random yeast two-hybrid screen of a lymphocyte cDNA library (using caspase-3 as the bait) found an interaction between caspase-3 and the regulatory subunit Aalpha of protein phosphatase 2A. This protein was found to be a substrate for caspase-3, but not caspase-1, and could compete effectively against either a protein or synthetic peptide substrate. In Jurkat cells induced to undergo apoptosis with anti-Fas antibody, protein phosphatase 2A (PP2A) activity increased 4.5-fold after 6 h. By 12 h, the regulatory Aalpha subunit could no longer be detected in cell lysates. There was no change in the amount of the catalytic subunit. The effects on PP2A could be prevented by the caspase family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone. The mitogen-activated protein (MAP) kinase pathway is regulated by PP2A. At 12 h after the addition of anti-Fas antibody, a decrease in the amount of the phosphorylated forms of MAP kinase was observed. Again, this loss of activated MAP kinase could be prevented by the addition of DEVD-cho or DEVD-fmk. These data are consistent with a pathway whereby induction of apoptosis activates caspase-3. This enzyme then cleaves the regulatory Aalpha subunit of PP2A, increasing its activity. These data show that the activated PP2A will then effect a change in the phosphorylation state of the cell. These data provide a link between the caspases and signal transduction pathways.

Apoptosis↗
PubMed1998

Modulation of ceramide-activated protein phosphatase 2A activity by low molecular weight aromatic compounds.

Protein phosphatase 2A (PP2A) is one of the most important and abundant serine/threonine phosphatases in mammalian tissues and plays a role in gene expression, cell division, and signal transduction. PP2A is activated by ceramide, which is produced by the hydrolysis of membrane sphingomyelin in response to a variety of stress-related stimuli. To further study the role of ceramide-mediated signal transduction in cellular processes such as senescence and apoptosis, we designed and synthesized a series of low molecular weight aromatic compounds, mainly of the isoquinolone and tetralone classes, and evaluated their ability to inhibit enzymes known to be activated by ceramide. Those enzymes studied were ceramide-activated protein kinase, protein kinase C zeta and PP2A. Of these, only PP2A was found to be inhibited. A few of the compounds inhibited both ceramide-activated as well as basal PP2A activity. In addition, several of the compounds activated PP2A by up to 300% above basal enzyme activity, but only in the presence of ceramide. Thus, modulation (both inhibition and activation) of the catatylic activity of ceramide-activated PP2A is demonstrated by certain low molecular weight aromatic compounds.

Blotting, Western↗
PubMed1998

An ordered Arabidopsis thaliana mitochondrial cDNA library on high-density filters allows rapid systematic analysis of plant gene expression: a pilot study.

The availability of the complete sequence of a genome allows a systematic analysis of its expression. Gene-specific variations of transcription levels and phenomena such as transcript processing and RNA editing require large numbers of clones to be examined. For the completely sequenced mitochondrial genome of Arabidopsis thaliana we adapted robot technology to identify and characterize expressed genes. A cDNA library of about 50,000 clones was constructed, robot-ordered into 384-well microtitre plates and spotted onto high-density filter membranes. These filters permit the isolation of large numbers of specific cDNA clones in a single hybridization step. The cox1, cox2 and cox3 genes were used to evaluate the feasibility and efficiency of this approach. A cluster of RNA editing sites observed outside the cox3 coding region identifies a novel reading frame orf95 in higher plants with significant similarity to a subunit of respiratory chain complex II.

Arabidopsis↗
PubMed1998

1995 assisted reproductive technology success rates: national summary and fertility clinic report.

Although ART offers important options for the treatment of infertility, the decision to use ART involves many factors in addition to success rates. Going through repeated ART cycles requires substantial commitments of time, effort, money, and emotional energy. Couples and individuals considering ART should carefully examine all related financial, psychologic, ethical, and medical issues before beginning treatment. They should also contact ART clinics to discuss their specific medical situation and potential for success using ART. The next published report will feature 1996 data and provide a listing of clinics that did not submit data. Eventually, the annual report will include information from all U.S. fertility clinics, not just those that are SART members.

Adult↗
PubMed1998

Development, validation, and interlaboratory comparison of an HMG-CoA reductase inhibition assay for quantitation of atorvastatin in plasma matrices.

An HMG-CoA reductase inhibition assay was developed and validated for quantitation of atorvastatin in human, dog, rat, and mouse plasma. Atorvastatin was isolated from plasma by protein precipitation. Rat-liver microsomes were used to provide the reductase enzyme. The method was validated by assaying calibration standards and quality controls in triplicate on each of the 3 days. A customized computer program was used for data calculation. Quantitation of the assay ranged from 0.36 to 16 ng/ml of atorvastatin in different plasma matrices. Assay precision and accuracy, based on the coefficient of variation and percent relative error, respectively, of quality controls were 10.4% to 14.5% and within +/- 6.25% in human; 4.89% to 10.6% (+/- 8.13%) in dog; 2.68% to 8.62% (+/- 5.00%) in rat; and 3.68% to 8.96% (+/- 5.38%) in mouse plasma. The method has been applied to pharmacokinetic studies of atorvastatin in human and toxicokinetic studies in dog, rat, and mouse after atorvastatin administration. Atorvastatin equivalent concentrations in a set of plasma samples from subjects receiving single and multiple doses of atorvastatin were determined by validated HMG-CoA reductase inhibition assays at four different laboratories. Results were compared using linear regression and concordance correlation statistical procedures. Good agreements among these data indicated that results from different laboratories with the same validated method can be used interchangeably.

Animals↗
PubMed1998

Lithium augmentation of venlafaxine in adolescent major depression.

OBJECTIVE: Studies on the pharmacotherapy of adolescent major depression are limited but suggest that the illness is often resistant to drug treatment. We aim to report the use of lithium augmentation of venlafaxine in two teenagers with major depression. CLINICAL PICTURE: Two 16-year-olds with major depression are described. TREATMENT: In both patients, the treatment involved trials of a selective serotonin reuptake inhibitor, venlafaxine, and psychotherapy preceded lithium augmentation of venlafaxine. The trial of venlafaxine alone had been short in one patient. OUTCOME: Combining lithium and venlafaxine was rapidly followed by marked improvement in mood and function. CONCLUSIONS: Augmentation of antidepressants should be considered in young depressed patients who fail to respond to adequate trials of new antidepressants alone.

Adolescent↗
PubMed1998

Attitudes of mental health personnel towards rating outcome.

Outcome measurement is becoming an integral part of health-care delivery. The attitudes of staff towards this development are important as they may influence ratings themselves, improve the design of measures and indicate how outcome assessment should be introduced into services. In this preliminary study, we surveyed staff from Central Sydney Mental Health Services who had taken part in a Commonwealth-funded project which had required them to rate patient outcome. The major concern expressed by respondents was that rating outcome was too time-consuming. More than half were not in favor of measuring outcome routinely even if it meant providing a better service to patients. The study methodology had limitations. However, if our findings are corroborated, attention will need to be paid to staff attitudes to outcome assessment so that ratings are more reliable and valid, properly inform treatment and meaningfully influence resource allocation.

Attitude of Health Personnel↗
PubMed1997

Increasing the ratio of PP2A core enzyme to holoenzyme inhibits Tat-stimulated HIV-1 transcription and virus production.

We demonstrated previously that PP2A exists in many cell types as two abundant forms: (1) holoenzyme composed of two regulatory subunits, A and B, and a catalytic subunit C; and (2) core enzyme consisting of the A and C subunits. These two forms have different substrate specificities. Since published data suggested that HIV-1 transcription may be regulated by a cellular protein phosphatase, it was of interest to determine whether changing the ratio between PP2A core and holoenzyme affects HIV-1 gene expression. This question was addressed by expression in COS cells of an N-terminal mutant of the A subunit, A delta 5, which binds the C but not the B subunit. This resulted in an increase in the amount of core enzyme and a decrease in the amount of holoenzyme concomitant with the expected change in phosphatase activity. Tat-stimulated transcription from the HIV-1 LTR was inhibited 5-fold by mutant A delta 5, whereas mRNA synthesis directed by the actin promoter was not affected. Furthermore, virus production in COS, HeLa, and Jurkat T cells was inhibited 45-, 5-, and 3-fold, respectively, by mutant A delta 5. These results demonstrate that the balance between PP2A holoenzyme and core enzyme is important for HIV-1 gene expression and virus production.

Animals↗
PubMed1997

HRX leukemic fusion proteins form a heterocomplex with the leukemia-associated protein SET and protein phosphatase 2A.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins. Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia. We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A.T hooks that have been shown to bind DNA. We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX. We have also found serine/threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates. Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A). Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A. These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.

Amino Acid Sequence↗
PubMed1997

Perioperative effects of alpha-stat versus pH-stat strategies for deep hypothermic cardiopulmonary bypass in infants.

OBJECTIVES: In a randomized, single-center trial, we compared perioperative outcomes in infants undergoing cardiac operations after use of the alpha-stat versus pH-stat strategy during deep hypothermic cardiopulmonary bypass. METHODS: Admission criteria included reparative cardiac surgery, age less than 9 months, birth weight 2.25 kg or more, and absence of associated congenital or acquired extracardiac disorders. RESULTS: Among the 182 infants in the study, diagnoses included D-transposition of the great arteries (n = 92), tetralogy of Fallot (n = 50), tetralogy of Fallot with pulmonary atresia (n = 6), ventricular septal defect (n = 20), truncus arteriosus (n = 8), complete atrioventricular canal (n = 4), and total anomalous pulmonary venous return (n = 2). Ninety patients were assigned to alpha-stat and 92 to pH-stat strategy. Early death occurred in four infants (2%), all in the alpha-stat group (p = 0.058). Postoperative electroencephalographic seizures occurred in five of 57 patients (9%) assigned to alpha-stat and one of 59 patients (2%) assigned to pH-stat strategy (p = 0.11). Clinical seizures occurred in four infants in the alpha-stat group (4%) and two infants in the pH-stat group (2%) (p = 0.44). First electroencephalographic activity returned sooner among infants randomized to pH-stat strategy (p = 0.03). Within the homogeneous D-transposition subgroup, those assigned to pH-stat tended to have a higher cardiac index despite a lower requirement for inotropic agents; less frequent postoperative acidosis (p = 0.02) and hypotension (p = 0.05); and shorter duration of mechanical ventilation (p = 0.01) and intensive care unit stay (p = 0.01). CONCLUSIONS: Use of the pH-stat strategy in infants undergoing deep hypothermic cardiopulmonary bypass was associated with lower postoperative morbidity, shorter recovery time to first electroencephalographic activity, and, in patients with D-transposition, shorter duration of intubation and intensive care unit stay. These data challenge the notion that alpha-stat management is a superior strategy for organ protection during reparative operations in infants using deep hypothermic cardiopulmonary bypass.

Acid-Base Equilibrium↗
PubMed1997

An epidemiological study of the use of ECT in adolescents.

OBJECTIVE: There is little knowledge about the use of electroconvulsive therapy (ECT) in adolescents. Given the prevalence and severity of psychiatric disorders in this age group, it is important to determine the frequency, indications, effectiveness, and side effects of ECT. METHOD: Persons younger than 19 years who received ECT between 1990 and 1996 in the Australian state of New South Wales were identified. Detailed information about diagnosis, treatment, and outcome was then obtained. RESULTS: Forty-two patients aged 14 to 18 years underwent a total of 49 courses comprising 450 ECTs (0.93% of all treatments given to all persons). Marked improvement or resolution of symptoms occurred in half of the completed courses. Mood disorders derived most benefit from ECT. Side effects were transient and minor. Prolonged seizures were observed in 0.4% of treatments. Comorbid personality disorder predicted poorer response, and the anesthetic propofol was associated with shorter seizures. CONCLUSIONS: Although ECT is an effective treatment for some mental disorders in adolescents and has few side effects, it is seldom used. Indications, response, and unwanted effects were similar to those observed in adults. The use of propofol may reduce the risk of prolonged seizures.

Adolescent↗
PubMed1997

Separation of PP2A core enzyme and holoenzyme with monoclonal antibodies against the regulatory A subunit: abundant expression of both forms in cells.

Protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit, C, and two regulatory subunits, A and B. The A subunit is rod shaped and consists of 15 nonidentical repeats. According to our previous model, the B subunit binds to repeats 1 through 10 and the C subunit binds to repeats 11 through 15 of the A subunit. Another form of PP2A, core enzyme, is composed only of subunits A and C. It is generally believed that core enzyme does not exist in cells but is an artifact of enzyme purification. To study the structure and relative abundance of different forms of PP2A, we generated monoclonal antibodies against the native A subunit. Two antibodies, 5H4 and 1A12, recognized epitopes in repeat 1 near the N terminus and immunoprecipitated free A subunit and core enzyme but not holoenzyme. Another antibody, 6G3, recognized an epitope in repeat 15 at the C terminus and precipitated only the free A subunit. Monoclonal antibodies against a peptide corresponding to the N-terminal 11 amino acids of the A alpha subunit (designated 6F9) precipitated free A subunit, core enzyme, and holoenzyme. 6F9, but not 5H4, recognized holoenzymes containing either B, B', or B" subunits. These results demonstrate that B subunits from three unrelated gene families all bind to repeat 1 of the A subunit, and the results confirm and extend our model of the holoenzyme. By sequential immunoprecipitations with 5H4 or 1A12 followed by 6F9, core enzyme and holoenzyme in cytoplasmic extracts from 10T1/2 cells were completely separated and they exhibited the expected specificities towards phosphorylase a and retinoblastoma peptide as substrates. Quantitative analysis showed that under conditions which minimized proteolysis and dissociation of holoenzyme, core enzyme represented at least one-third of the total PP2A. We conclude that core enzyme is an abundant form in cells rather than an artifact of isolation. The biological implications of this finding are discussed.

Animals↗