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K Maxwell

Publications and source records attributed to K Maxwell.

At least 19 recordsLinked to original sources
PubMed2001

Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme.

The hairpin ribozyme in its natural context consists of two loops in RNA duplexes that are connected as arms of a four-way helical junction. Magnesium ions induce folding into the active conformation in which the two loops are in proximity. In this study, we have investigated nucleotides that are important to this folding process. We have analyzed the folding in terms of the cooperativity and apparent affinity for magnesium ions as a function of changes in base sequence and functional groups, using fluorescence resonance energy transfer. Our results suggest that the interaction between the loops is the sum of a number of component interactions. Some sequence variants such as A10U, G+1A, and C25U exhibit loss of cooperativity and reduced affinity of apparent magnesium ion binding. These variants are also very impaired in ribozyme cleavage activity. Nucleotides A10, G+1, and C25 thus appear to be essential in creating the conformational environment necessary for ion binding. The double variant G+1A/C25U exhibits a marked recovery of both folding and catalytic activity compared to either individual variant, consistent with the proposal of a triple-base interaction among A9, G+1, and C25 [Pinard, R., Lambert, D., Walter, N. G., Heckman, J. E., Major, F., and Burke, J. M. (1999) Biochemistry 38, 16035-16039]. However, substitution of A9 leads to relatively small changes in folding properties and cleavage activity, and the double variant G+1DAP/C25U (DAP is 2,6-diaminopurine), which could form an isosteric triple-base interaction, exhibits folding and cleavage activities that are both very impaired compared to those of the natural sequence. Our results indicate an important role for a Watson--Crick base pair between G+1 and C25; this may be buttressed by an interaction with A9, but the loss of this has less significant consequences for folding. 2'-Deoxyribose substitution leads to folding with reduced magnesium ion affinity in the following order: unmodified RNA > dA9 > dA10 > dC25 approximately dA10 plus dC25. The results are interpreted in terms of an interaction between the ribose ring of C25 and the ribose and base of A10, in agreement with the proposal of Ryder and Strobel [Ryder, S. P., and Strobel, S. A. (1999) J. Mol. Biol. 291, 295-311]. In general, there is a correlation between the ability to undergo ion-induced folding and the rate of ribozyme cleavage. An exception to this is provided by G8, for which substitution with uridine leads to severe impairment of cleavage but folding characteristics that are virtually unaltered from those of the natural species. This is consistent with a direct role for the nucleobase of G8 in the chemistry of cleavage.

2-Aminopurine↗
PubMed2000

Glucose uptake increases relative to oxygen consumption during short-term hibernation.

BACKGROUND: Although glucose uptake is increased in chronically hypoperfused, viable myocardium, the dynamic changes in glucose uptake relative to oxygen consumption in "short-term" models of hibernation have not been fully explored. METHODS: 14 anesthetized swine were instrumented with an hydraulic occluder and flow probe on the proximal LAD artery. Blood flow was reduced approximately 30% for 1 hour. Myocardial blood flow and uptake of oxygen, free fatty acids, glucose and lactate were determined in the LAD region at baseline and at 10, 30, and 60 minutes of ischemia. Transmural biopsies for ATP and creatine phosphate (CP) were obtained in the LAD region prior to and at 15 and 45 minutes of ischemia. In 5 animals, glycogen was assayed at baseline and at the end of 60 minutes of ischemia. RESULTS: In the LAD region, myocardial oxygen consumption was reduced from 2.06 +/- 0.16 micromol/min/gram to 1.46 +/- 0.13 micromol/min/gram (P < 0.05). By 15 minutes of ischemia, transmural creatine phosphate fell from 7.48 +/- 0.76 micromol/g-wet weight at baseline to 6.19 +/- 0.32 micromol/g-wet weight (P < 0.05) but normalized by 45 minutes of ischemia (7.39 +/- 0.56 micromol/g-wet weight; NS). Between 10 and 60 minutes of constant flow reduction, glucose uptake as a percentage of MVO2 increased from 3 +/- 2% to 10 +/- 2% (P < 0.05) while lactate uptake increased from -9 +/- 9% to -1 +/- 2% (P < 0.05). Glycogen decreased from 27.8 +/- 3.7 at baseline to 16.9 +/- 1.2 micromol/g-wet weight at end-ischemia. CONCLUSIONS: In this model of short-term hibernation, glucose and lactate uptake increase relative to oxygen consumption during sustained ischemia, and temporally coincide with the recovery of bioenergetics. The findings are consistent with the notion that glycolytically derived ATP is important for the maintainance of energy supply during sustained ischemia.

Animals↗
PubMed2000

The folding of the hairpin ribozyme: dependence on the loops and the junction.

In its natural context, the hairpin ribozyme is constructed around a four-way helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. In the present study we have compared the folding of this form of the ribozyme and subspecies lacking either the loops or the helical junction using fluorescence resonance energy transfer. The complete ribozyme as a four-way junction folds into an antiparallel structure by the cooperative binding of magnesium ions, requiring 20-40 microM for half-maximal extent of folding ([Mg2+]1/2) and a Hill coefficient n = 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n = 1) at a higher magnesium ion concentration ([Mg2+]1/2 = 3 mM). Introduction of a G + 1A mutation into loop A of the ribozyme results in a species with very similar folding to the simple junction, and complete loss of ribozyme activity. Removal of the junction from the ribozyme, replacing it either with a strand break (serving as a hinge) or a GC5 bulge, results in greatly impaired folding, with [Mg2+]1/2 > 20 mM. The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop-loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in the natural RNA that facilitates the folding of the ribozyme into the active form.

Base Sequence↗
PubMed2000

Chlorophyll fluorescence--a practical guide.

Chlorophyll fluorescence analysis has become one of the most powerful and widely used techniques available to plant physiologists and ecophysiologists. This review aims to provide an introduction for the novice into the methodology and applications of chlorophyll fluorescence. After a brief introduction into the theoretical background of the technique, the methodology and some of the technical pitfalls that can be encountered are explained. A selection of examples is then used to illustrate the types of information that fluorescence can provide.

Chlorophyll↗
PubMed1999

Experiences of hospital care and treatment seeking for pain from sickle cell disease: qualitative study.

OBJECTIVE: To investigate how sociocultural factors influence management of pain from sickle cell disease by comparing the experiences of those who usually manage their pain at home with those who are more frequently admitted to hospital for management of their pain. DESIGN: Qualitative analysis of semistructured individual interviews and focus group discussions. PARTICIPANTS: 57 participants with genotype SS or S/beta-thal (44 subjects) or SC (9) (4 were unknown). 40 participants took part in focus groups, six took part in both focus groups and interviews, and nine were interviewed only. Participants were allocated to focus groups according to number of hospital admissions for painful crisis management during the previous year, ethnic origin, and sex. RESULTS: The relation between patients with sickle cell disease and hospital services is one of several major non-clinical dimensions shaping experiences of pain management and behaviour for seeking health care. Experiences of hospital care show a range of interrelated themes, which are common to most participants across variables of sex, ethnicity, and hospital attended: mistrust of patients with sickle cell disease; stigmatisation; excessive control (including both over- and undertreatment of pain); and neglect. Individuals respond to the challenge of negotiating care with various strategies. Patients with sickle cell disease who are frequently admitted to hospital may try to develop long term relationships with their carers, may become passive or aggressive in their interactions with health professionals, or may regularly attend different hospitals. Those individuals who usually manage their pain at home express a strong sense of self responsibility for their management of pain and advocate self education, assertiveness, and resistance as strategies towards hospital services. CONCLUSIONS: The current organisation and delivery of management of pain for sickle cell crisis discourage self reliance and encourage hospital dependence. Models of care should recognise the chronic nature of sickle cell disorders and prioritise patients' involvement in their care.

Adult↗
PubMed1999

Risperidone-induced hepatotoxicity in children and adolescents? A chart review study.

Risperidone is an atypical antipsychotic drug that has been used in the treatment of numerous psychiatric disorders in children and adolescents. The question of whether risperidone-induced weight gain is associated with steatohepatitis has recently been raised. The purpose of this chart review was to ascertain: (1) the rate of liver dysfunction observed during risperidone treatment in children and adolescents; and (2) the clinical factors associated with liver dysfunction. For purposes of this chart review study, abnormal liver function was defined by serum transaminase or bilirubin values falling outside the normal laboratory ranges. Chart reviews were completed on 38 youths with ages ranging from 5-17 years with a variety of psychiatric diagnoses. The mean length of risperidone treatment was 15.2 months at a mean dose of 2.5 mg/day. It was found that 37 of the 38 youths treated with risperidone had no liver enzyme abnormalities at the end of study. One subject had an alanine aminotransferase (ALT) level of 46 U/L which was 7 U/L above the upper limit of normal for this laboratory test. This isolated value was not considered clinically significant. These data were noted in spite of weight gain and the use of numerous concomitant psychotropic medications. These findings suggest that risperidone in short term treatment does not commonly lead to evidence of abnormal liver function at therapeutic doses in children and adolescents. Larger-scale, prospective studies are needed in order to confirm these findings.

Adolescent↗
PubMed1999

Chloroplast acclimation in leaves of Guzmania monostachia in response to high light.

Acclimation of leaves to high light (HL; 650 micromol m(-2) s(-1)) was investigated in the long-lived epiphytic bromeliad Guzmania monostachia and compared with plants maintained under low light (LL; 50 micromol m(-2) s(-1)). Despite a 60% decrease in total chlorophyll in HL-grown plants, the chlorophyll a/b ratio remained stable. Additionally, chloroplasts from HL-grown plants had a much lower thylakoid content and reduced granal stacking. Immunofluorescent labeling techniques were used to quantify the level of photosynthetic polypeptides. HL-grown plants had 30% to 40% of the content observed in LL-grown plants for the light-harvesting complex associated with photosystems I and II, the 33-kD photosystem II polypeptide, and Rubisco. These results were verified using conventional biochemical techniques, which revealed a comparable 60% decrease in Rubisco and total soluble protein. When expressed on a chlorophyll basis, the amount of protein and Rubisco was constant for HL- and LL-grown plants. Acclimation to HL involves a tightly coordinated adjustment of photosynthesis, indicating a highly regulated decrease in the number of photosynthetic units manifested at the level of the content of light-harvesting and electron transport components, the amount of Rubisco, and the induction of Crassulacean acid metabolism. This response occurs in mature leaves and may represent a strategy that is optimal for the resource-limited epiphytic niche.

Chlorophyll↗
PubMed1999

Modulation of Rubisco Activity during the Diurnal Phases of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana.

The regulation of Rubisco activity was investigated under high, constant photosynthetic photon flux density during the diurnal phases of Crassulacean acid metabolism in Kalanchoë daigremontiana Hamet et Perr. During phase I, a significant period of nocturnal, C(4)-mediated CO(2) fixation was observed, with the generated malic acid being decarboxylated the following day (phase III). Two periods of daytime atmospheric CO(2) fixation occurred at the beginning (phase II, C(4)-C(3) carboxylation) and end (phase IV, C(3)-C(4) carboxylation) of the day. During the 1st h of the photoperiod, when phosphoenolpyruvate carboxylase was still active, the highest rates of atmospheric CO(2) uptake were observed, coincident with the lowest rates of electron transport and minimal Rubisco activity. Over the next 1 to 2 h of phase II, carbamylation increased rapidly during an initial period of decarboxylation. Maximal carbamylation (70%-80%) was reached 2 h into phase III and was maintained under conditions of elevated CO(2) resulting from malic acid decarboxylation. Initial and total Rubisco activity increased throughout phase III, with maximal activity achieved 9 h into the photoperiod at the beginning of phase IV, as atmospheric CO(2) uptake recommenced. We suggest that the increased enzyme activity supports assimilation under CO(2)-limited conditions at the start of phase IV. The data indicate that Rubisco activity is modulated in-line with intracellular CO(2) supply during the daytime phases of Crassulacean acid metabolism.

Journal Article↗
PubMed1999

Functional role of ionic regulation of Na+/Ca2+ exchange assessed in transgenic mouse hearts.

Na+/Ca2+ exchange is the primary mechanism mediating Ca2+ efflux from cardiac myocytes during diastole and, thus, can prominently influence contractile force. In addition to transporting Na+ and Ca2+, the exchanger is also regulated by these ions. Although structure-function studies have identified protein regions of the exchanger subserving these regulatory processes, their physiological importance is unknown. In this study, we examined the electrophysiological and mechanical consequences of cardiospecific overexpression of the canine cardiac exchanger NCX1.1 and a deletion mutant of NCX1.1 (Delta680-685), devoid of intracellular Na+ (Na+i)- and Ca2+ (Ca2+i)- dependent regulatory properties, in transgenic mice. Using the giant excised patch-clamp technique, normal ionic regulation was observed in membrane patches from cardiomyocytes isolated from control and transgenic mice overexpressing NCX1.1. In contrast, ionic regulation was nearly abolished in mice overexpressing Delta680-685, indicating that the native regulatory processes could be overwhelmed by expression of the transgene. To address the physiological consequences of ionic regulation of the Na+/Ca2+ exchanger, we examined postrest force development in papillary muscles from NCX1.1 and Delta680-685 transgenic mice. Postrest potentiation was found to be substantially greater in Delta680-685 than in NCX1.1 transgenic mice, supporting the notion that ionic regulation of Na+/Ca2+ exchange plays a significant functional role in cardiac contractile properties.

Animals↗
PubMed1998

Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites.

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+i- and Ca2+i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that microM levels of Ca2+i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+i binding site of CALX1. 1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+i to inhibit Na+-Ca2+ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta310-313) of Na+i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+i. This result indicates that the regulatory Ca2+i binding site of NCX1.1 retains function in a CALX1. 1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+i binding signal.

Amino Acid Sequence↗
PubMed1998

Biology and function of the reversible sulfation pathway catalysed by human sulfotransferases and sulfatases.

Sulfation and sulfate conjugate hydrolysis play an important role in metabolism, and are catalysed by members of the sulfotransferase and sulfatase enzyme super-families. In general, sulfation is a deactivating, detoxication pathway, but for some chemicals the sulfate conjugates are much more reactive than the parent compound. The range of compounds which are sulfated is enormous, yet we still understand relatively little of the function of this pathway. This review summarises current knowledge of the sulfation system and the enzymes involved, and illustrates how heterologous expression of sulfotransferases (SULTs) and sulfatases is aiding our appreciation of the properties of these important proteins. The role of sulfation in the bioactivation of procarcinogens and promutagens is discussed, and new data on the inhibition of the sulfotransferase(s) involved by common dietary components such as tea and coffee are presented. The genetic and environmental factors which are known to influence the activity and expression of human SULTs and sulfatases are also reviewed.

Amino Acid Sequence↗
PubMed1998

Laser-cured fibrinogen glue to repair bleb leaks in rabbits.

OBJECTIVE: To determine whether laser-cured fibrinogen glue can close bleb leaks in rabbits. METHODS: Full-thickness filtration surgery with intraoperative mitomycin and a sutured limbus-based conjunctival flap was performed in 1 eye each of 19 New Zealand albino rabbits. On the second postoperative day, a 2- to 3-mm hole was made in the bleb. In 9 rabbits, the hole was glued using fibrinogen glue with indocyanine green dye added. The glue was "cured" with a diode laser. Eyes that had been glued and developed a subsequent leak had the glue reapplied on the day the leak was detected. RESULTS: The glue remained on the conjunctiva for an average (mean+/-SD) of 1.9+/-1.8 days (range, 0-5 days). The last day of bleb leak for the rabbits with glued eyes was 1.6+/-2.4 days; for the control rabbits, it was 8.0+/-4.4 days (P=.001, Mann-Whitney U test). CONCLUSION: Laser-cured fibrinogen glue is effective in closing bleb leaks in rabbits.

Animals↗
PubMed1998

Endoscopic creation of reflux in the pig.

Vesicoureteral reflux (VUR) in the animal model for experimental purposes can be created either by open transvesical or endoscopic techniques. The concept of reflux creation is the same for both techniques: incision of the roof of the intramural portion of the ureter at the 12 o'clock position. The open method has the disadvantages of requiring a cystotomy and a lengthy healing period prior to initiating a study, thereby incurring additional expense and the problem of introducing several confounding factors. The open method is unreliable because of the resolution of reflux over time. Herein, we present a simple transurethral endoscopic technique for creating VUR in pigs. This technique was successful in producing persistent Grade II or III reflux in 94% of the incised ureters.

Animals↗
PubMed1998

Not a 'proper' solution? The gap between professional guidelines and users' views about the safety of using emergency contraception.

OBJECTIVES: As a form of contraception which is used after sex, emergency contraception occupies a singular place in the birth control repertoire. The relatively high UK incidence of pregnancy terminations and of teenage pregnancy, combined with the recognition that much early sex remains unplanned and unprotected, has led to calls for better access to emergency contraceptive methods. In this study a combination of self-completion questionnaires and semi-structured interviews was used to explore views of emergency contraception among women who were using the method. METHODS: Five hundred and ten women attending two family planning clinics in Oxford and London completed a questionnaire in the waiting room and 53 women who were attending for emergency contraception took part in semi-structured interviews. RESULTS: The view, presented in recently published UK guidelines, that emergency contraception is a reliable method and not dangerous to repeat, was not shared by the respondents. The rationale for and sources of women's concerns about the strength of the dose of hormonal emergency contraception and the nature of side-effects are explored.

Adolescent↗
PubMed1997

Cytokine expression in otitis media with effusion. Tumor necrosis factor soluble receptor.

BACKGROUND: A variety of cytokines, such as interleukin 1 and tumor necrosis factor (TNF), appear to play a key role in the initiation and maintenance of the inflammatory response in a variety of diseases, including chronic otitis media with effusion (COME). Recently, cytokine inhibitors have been isolated and are presumed to regulate the proinflammatory effects of these cytokines. This has lead us to hypothesize that the chronic inflammation seen in COME is the result of an imbalance in the ratio of proinflammatory cytokines and inhibitors that favors the proinflammatory cytokines. DESIGN: Middle ear effusions were evaluated for the cytokine TNF-alpha and the inhibitor TNF soluble receptor (TNFsolR) using a combination of enzyme-linked immunosorbent assays and radioactive immunoassays. We determined the presence and quantified the amounts of TNF-alpha and TNFsolR and correlated this result with clinical parameters in patients with COME. SETTING: University hospital. PATIENTS: To test this hypothesis, 35 middle ear effusions were obtained from 29 children aged 2 to 102 months (mean, 39.6 months) undergoing tympanostomy tube insertion for COME. OUTCOME: Children were followed up for at least 12 months postoperatively and the number of subsequent episodes of otitis media correlated with cytokine levels. RESULTS: The TNF-alpha and TNFsolR were present in 83% and 91% of effusions, respectively. The TNF-alpha mean (+/-SEM) cytokine values were 110.6 +/- 32.1 pg/mg of total protein, and 12886 +/- 3108 pg/mg total protein for TNFsolR. Analysis of correlations in a univariate model revealed a statistically significant relationship for TNF-alpha correlated with TNFsolR (R2 = 0.463; P < .001). The TNF-alpha levels were significantly lower for children with multiple tube insertions (P = .02). Higher levels of TNF-alpha were noted in those children who subsequently developed episodes of otitis media after tube placement (P = .02). The ratio of TNF-alpha to TNFsolR, the mean (+/-SEM) inhibitor index was 430 +/- 220 U. CONCLUSIONS: Our data support the hypothesis that cytokines and their inhibitors are present in a large number of middle ear effusions and in part are likely important in the regulation of inflammatory processes in COME.

Analysis of Variance↗
PubMed1997

High-dose pyridoxine and magnesium administration in children with autistic disorder: an absence of salutary effects in a double-blind, placebo-controlled study.

Several reports have described salutary effects such as decreased physical aggression and improved social responsiveness being associated with the administration of high doses of pyridoxine and magnesium (HDPM) in open-labeled and controlled studies of patients with autism. Despite this fact, this intervention remains controversial. A 10-week double-blind, placebo-controlled trial was undertaken to examine both the efficacy and safety of HDPM in autism. Twelve patients were enrolled, and 10 patients (mean age 6 years 3 months) were able to complete the study. HDPM at an average dose of 638.9 mg of pyridoxine and 216.3 mg of magnesium oxide was ineffective in ameliorating autistic behaviors as assessed by the Children's Psychiatric Rating Scale (CPRS), the Clinical Global Impression Scale, and the NIMH Global Obsessive Compulsive Scale. Furthermore, no clinically significant side effects were noted during HDPM administration. A trend for a transient change on the CPRS was found that was possibly due to a placebo response. This study raises doubts about the clinical effectiveness of HDPM in autistic disorder.

Adolescent↗