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Biomedical subjects

K King

Publications and source records attributed to K King.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cell-surface perturbations of the epidermal growth factor and vascular endothelial growth factor receptors by phosphorothioate oligodeoxynucleotides.

Antisense oligodeoxynucleotides offer potential as therapeutic agents to inhibit gene expression. Recent evidence indicates that oligodeoxynucleotides designed to target specific nucleic acid sequences can interact nonspecifically with proteins. This report describes the interactive capabilities of phosphorothioate oligodeoxynucleotides of defined sequence and length with two essential protein tyrosine receptors, flk-1 and epidermal growth factor receptor (EGFR), and their effects on receptor signaling in a transfected and tumor cell line, respectively. Phosphorothioate oligodeoxynucleotides bound to the cell surface, as demonstrated by fluorescence-activated cell-sorter analyses (FACS), and perturbed receptor activation in the presence and absence of cognate ligands, EGF (EGFR) and vascular endothelial growth factor (flk-1), in phosphorylation assays. Certain phosphorothioate oligodeoxynucleotides interacted relatively selectively with flk-1 and partially blocked the binding of specific anti-receptor monoclonal antibodies to target sites. They stimulated EGFR phosphorylation in the absence of EGF but antagonized ligand-mediated activation of EGFR and flk-1. In vivo studies showed that a nonspecific phosphorothioate oligodeoxynucleotide suppressed the growth of glioblastoma in a mouse model of tumorigenesis. These results emphasize the capacity of phosphorothioate oligodeoxynucleotides to interact with cells in a sequence-selective nonantisense manner, while associating with cellular membrane proteins in ways that can inhibit cellular metabolic activities.

3T3 Cells↗
PubMed1997

Antibiotics: effect on cryopreserved-thawed human sperm motility in vitro.

OBJECTIVE: To analyze the motility and fertilizing capacity of sperm treated with different antibiotics. DESIGN: Prospective comparative study. SETTING: Clinical and academic research environment. PATIENT(S): Pooled cryopreserved donor sperm (n = 14). INTERVENTION(S): Sperm were washed with Percoll and resuspended in HEPES-buffered human tubal fluid medium containing either amoxicillin, ofloxacin, ciprofloxacin hydrochloride, nitrofurantoin monohydrate, doxycycline hyclate, cefuroxime axetil, or control medium. MAIN OUTCOME MEASURE(S): Sperm kinematic and fertilizing parameters. RESULT(S): Sperm hyperactivation was decreased in physiologic concentrations of ciprofloxacin hydrochloride and doxycycline hyclate over the course of 48 hours. At pharmacologic concentrations, ciprofloxacin hydrochloride, cefuroxime axetil, and nitrofurantoin monohydrate adversely affected motility with decreased rapid progression. Cessation of motility occurred in cefuroxime axetil and nitrofurantoin monohydrate. Sperm hyperactivation was also absent. Cefuroxime axetil decreased the percentage of intact acrosomes. In contrast, physiologic doses of ciprofloxacin hydrochloride or ofloxacin enhanced sperm fertilizing capacity. CONCLUSION(S): Ciprofloxacin affected hyperactivation by altering membrane properties, whereas doxycycline inhibited the capacitation process. Cessation of motility in cefuroxime axetil was linked to disrupted sperm head membranes. Sperm motility and fertilizing capacity were decreased in nitrofurantoin because of decreased metabolism. The positive effect of ofloxacin on fertilizing capacity did not involve changes in acrosome.

Analysis of Variance↗
PubMed1997

Supporting multi-level medical education with knowledge-based systems.

Knowledge-based systems for medicine have enjoyed minimal success in developing countries as end-user systems. The reasons for this are complex. As funding agencies understandably tend to err on the side of caution, and knowledge-based systems are still (despite an almost 40 year history) seen as a new and untried technology, few have been implemented. Of those which have, most are inappropriately simple and thus do not fit in with the real-life clinical environment. In contrast to the sophisticated systems in use in developed countries which reflect a mature technology, the use of knowledge-based systems in medicine in developing countries has primarily revolved around simple 'expert' systems, where the program functions more as a 'guru' than as a support function. We propose the more appropriate use of these systems as educational tools in medicine. In this discussion paper we describe a multi-level programme to support medical education, focusing on patient information systems involving natural language generation, decision-support systems as educational aids for primary health-care workers and model-based reasoning tools which allow exploratory learning for physicians in training. Throughout this paper we refer to Knowledge-Based Medical Education Systems as KBMES.

Artificial Intelligence↗
PubMed1996

Phosphatidylinositol 4,5-bisphosphate binding to the pleckstrin homology domain of phospholipase C-delta1 enhances enzyme activity.

The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some co-complexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-delta1 (PLCdelta1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in a dose-dependent manner with an EC50 = 1 microM (0.3 mol%), up to 9-fold higher when 5 microM (1.5 mol%) of PIP2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 microM of PI with a molar ratio of PI:PS:PC = 1:5:5). Stimulation was specific for PIP2, since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited by inositol 1,4, 5-triphosphate (IP3) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLCdelta1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP2-mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP2. Analysis of PIP2-stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (Ks) of PLCdelta1 for the PI/PS/PC vesicles was reduced from 558 microM to 53 microM, and the interfacial Michaelis constant (Km) was reduced from 0.21 to 0.06 by PIP2. The maximum rate of PI hydrolysis (Vmax) was not affected by PIP2. These results demonstrate that a major function of the PH domain of PLCdelta1 is to modulate enzyme activity. Further, our results identify PIP2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP2.

Binding Sites↗
PubMed1996

Positive charge at position 549 is essential for phosphatidylinositol 4,5-bisphosphate-hydrolyzing but not phosphatidylinositol-hydrolyzing activities of human phospholipase C delta1.

Point mutagenesis, phosphatidylinositol (PI), and phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis assays and equilibrium centrifugation PIP2 assays were used to study the functional roles of four highly conserved arginine residues in the Y region of human phospholipase C delta1 (PLCdelta1) (Arg-527, -549, -556, -701). Most of the mutant enzymes were either partially defective or fully active in their abilities to catalyze the hydrolysis of PI or PIP2. However, upon substitution of Arg-549 by glycine or histidine, the mutant enzyme was defective in its ability to catalyze the hydrolysis of PIP2, but it is still able to hydrolyze PI. Replacing Arg-549 with lysine had little effect on the level of PI and PIP2 hydrolytic activities of the mutant enzyme. The residual PIP2 hydrolyzing activity of R549H is highly dependent on pH. R549H showed 5-10% of the PIP2-hydrolyzing activity of the native enzyme between pH 5 and 7 and nondetectable PIP2-hydrolyzing activity at pH 8. The PIP2-hydrolyzing activity of R549G was not detectable at all pH values. Kinetic analysis of PLCdelta1-catalyzed PIP2 hydrolysis revealed that the micellar dissociation constant Ks and interfacial Michaelis constant Km were similar in the native, R549K, and R549H enzymes; but the specific activity at the saturated substrate mole fraction and infinite level of substrate (Vmax) of the R549H mutant were reduced by a factor of 15. PIP2 competitively inhibits the native enzyme to hydrolyze PI at both pH 7 and 8. However, PIP2 inhibits R549H only at pH 7.0 and does not inhibit R549G at either pH. Taken together, these results suggest that positive charge at position 549 of PLCdelta1 protein is essential for the enzyme to recognize and catalyze the hydrolysis of PIP2 but not PI.

Calcium↗
PubMed1996

Overexpression, purification, and generation of a thermostable variant of Zymomonas mobilis fructokinase.

The gene encoding fructokinase (EC 2.7.1.4) from Zymomonas mobilis has been expressed at high level in Escherichia coli by modifying the ribosome binding site using the polymerase chain reaction. A simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. Using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking the 30 min half-life from 70.1 to 72.4 degrees C. The purified thermostable variant had the same specific activity as the wild type. Sequencing of the variant indicated that only one amino acid was changed, with Ser 69 becoming Ala. Searches of the mutant libraries for variants that were (a) active with glucose or (b) had reduced inhibition by glucose were unsuccessful.

DNA Primers↗
PubMed1996

Economic value of gemcitabine compared to cisplatin and etoposide in non-small cell lung cancer.

Although chemotherapy costs have not been highlighted traditionally, there is increasing pressure to demonstrate the value of new treatments within the health care budget. Pharmaceutical companies are assessing the economic value of their products before launch. Gemcitabine is a nucleoside analogue developed for use in solid tumours. The purpose of this model was to investigate the clinical outcomes and potential cost savings for gemcitabine used as monotherapy compared to cisplatin and etoposide combination therapy in late stage non-small cell lung cancer (NSCLC), in a palliative (as opposed to aggressive) chemotherapy setting. Gemcitabine treatment data were taken from a large NSCLC study and data from retrospective chart reviews identified through the National Oncology Data Base. The model population and effectiveness of the two regimens were judged to be similar, except for baseline performance status. If drug costs were not included, the probability distribution resulting from the simulation showed median cost savings per cycle ranging from $US 1504 to $US 7425, with a medium value of $US 2154. The model suggested that gemcitabine would result in cost savings per cycle more than 90% of the time. Outpatient versus inpatient drug administrations accounted for the majority of potential cost savings. Most of the remaining cost savings were attributable to the difference in febrile neutropenia and antiemetic use. This economic model showed susbstantial savings if gemcitabine was used instead of cisplatin and etoposide combination therapy in the United States' community care setting. Some savings would be realized even if the location of treatment for both regimens was mostly outpatient. Assessment of the product's economic value before launch has assisted in our understanding of the potential areas of cost savings for gemcitabine and has guided us in the design of prospective randomized studies which included pharmacoeconomic endpoints.

Antimetabolites, Antineoplastic↗
PubMed1995

Are health undertakings effective in the follow-up of migrants for tuberculosis?

OBJECTIVE: To determine the rate of compliance of migrants issued with a health undertaking for tuberculosis requiring them to report within one month after arrival in Australia. DESIGN: Compliance was monitored prospectively between 1 November 1992 and 31 October 1993. SETTING: The Australian Government Health Service in Melbourne. PARTICIPANTS: 1660 migrants with a health undertaking for tuberculosis. MAIN OUTCOME MEASURE: Compliance with the undertaking (making initial contact and presenting for examination). RESULTS: The overall compliance rate was 58%, but 89% of those who made contact complied with their undertakings. There were significant differences in the compliance rates according to country of application (P < 0.001). It was estimated that about 150 people a year who require treatment for tuberculosis could be failing to comply with a health undertaking, eight of whom may require a full course of antituberculous chemotherapy. CONCLUSIONS: The administration of the current system of health undertakings for tuberculosis for migrants needs to be strengthened to increase compliance.

Australia↗
PubMed1995

Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.

In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

Amino Acid Sequence↗
PubMed1995

Differential homogenization and amplification of two satellite DNAs in the genus Cucurbita (Cucurbitaceae).

Two different satellite DNAs exist in the genus Cucurbita which are different with respect to repeat length (350 bp and 170 bp), array size, and sequence homogenization. Whereas the 350-bp satellite DNA is prominent and very homogeneous in all species investigated except for C. maxima and C. lundelliana, the 170-bp satellite is rather evenly distributed in all species. In C. maxima and C. lundelliana the 350-bp satellite is present only in small amounts, but detectable by the sensitive PCR method. These repeats are also very homogeneous, reflecting a silent stage of satellite DNA. In contrast, the 170-bp satellite DNA is intra- and interspecifically heterogeneous. It is striking that the species with no detectable amount of 350-bp satellite contain 170-bp satellite DNA clusters with the highest degree of homogeneity. The evolution of satellite DNA repeats within cultivated and wild species in the genus Cucurbita is elucidated using the sequence data of both satellite DNAs from all species investigated. The value of satellite DNA for phylogenetic analysis between closely related species is discussed.

Base Sequence↗
PubMed1995

IL-7 enhancement of antigen-driven activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp).

CD8+ cytotoxic T lymphocytes are an important component in the immunologic control of human viral diseases. IL-7, a stromal cell-derived cytokine, has been demonstrated to enhance both anti-tumour and anti-viral CTL as well as lymphokine-activated killer (LAK) activity. We studied the ability of IL-7 to support the activation and the growth of in vitro antigen-specific CTL precursors (CTLp) present in peripheral blood mononuclear cells (PBMC) from HIV-infected patients. Results from these studies demonstrate that inclusion of IL-7 in a vaccinia/HIV-1 vector-based stimulation strategy greatly augmented overall CTL reactivities, whereas addition of IL-7 to unstimulated cultures failed to induce any significant anti-viral cytolytic activity. In four of six patients, HIV-specific lytic activities were significantly higher in cultures stimulated with antigen plus IL-7 compared with in vitro stimulation (IVS) with antigen alone. Cytotoxic activity was principally mediated by CD8+ effector cells, and CD3+/CD8+ cell expansion was increased by 2.7-fold in the presence of IL-7. In PBMC from seronegative donors, IL-7 enhanced anti-vaccinia CTL activities with less effect on cell proliferation. Furthermore, anti-gag CTL frequencies determined by limiting dilution analysis were increased by 2- and 10-fold in two asymptomatic patients following IVS plus IL-7 compared with antigen stimulation alone. Cytofluorimetric analysis revealed that IL-7 preferentially expanded CD8 memory cells (CD45RO+) and CD8+ lymphocytes expressing activation molecules. IL-7 was also able to support the growth of CD4+ lymphocytes, while having no effect on natural killer (NK)/K lymphocytes. Taken together, these data suggest that IL-7 acts cooperatively with the antigen supporting in vitro maturation of CTLp into functional cytotoxic effectors. Thus IL-7 may be an important biologic entity to consider as part of future immune-based therapies in which ex vivo expansion of antigen-driven CTL is an important determinant.

Animals↗
PubMed1995

Acute hypertension increases norepinephrine release in the anterior hypothalamic area.

Neurons in the anterior hypothalamic area play an important role in NaCl-sensitive hypertension in spontaneously hypertensive rats, and previous studies have suggested that baroreceptor feedback modifies the activity of these neurons. To test the hypothesis that the release of norepinephrine in the anterior hypothalamic area is modified by arterial baroreceptor reflex feedback and that this reflex release is disturbed in spontaneously hypertensive rats on a high NaCl diet, we used the push-pull technique to measure the release of the norepinephrine metabolite 3-methoxy-4-hydroxy-phenylglycol in the anterior hypothalamic area. Seven-week-old male spontaneously hypertensive and normotensive Wistar-Kyoto rats were placed on a high (8%) or a basal (1%) NaCl diet for 2 weeks. The high NaCl diet elevated mean arterial pressure and greatly reduced basal norepinephrine metabolite levels in the anterior hypothalamic area of the spontaneously hypertensive (but not the control) rats (305 +/- 32 pg/10 min in the rats consuming 1% NaCl and 93 +/- 9 pg/10 min in the rats consuming 8% NaCl). An infusion of tramazoline (an imidizoline that causes long-lasting hypertension) that increased arterial pressure by 25 mm Hg elevated anterior hypothalamic area norepinephrine metabolite concentrations significantly more in the spontaneously hypertensive rats on the 1% NaCl diet (to 392 +/- 46 pg/10 min) than in those on the 8% NaCl diet (to 113 +/- 18 pg/10 min). In contrast, in Wistar-Kyoto rats the tramazoline-induced increase in arterial pressure elevated anterior hypothalamic area norepinephrine metabolite concentrations slightly more in rats on the 8% NaCl diet than in those on the 1% NaCl diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗