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K Middleton

Publications and source records attributed to K Middleton.

17 recordsLinked to original sources
PubMed2000

Optimization of storage stability of lyophilized actin using combinations of disaccharides and dextran.

The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting T(g) of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and T(g) by freeze-drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in T(g) observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability.

Actins↗
PubMed2000

Characterisation of extracellular polysaccharides from suspension cultures of members of the poaceae.

Microscopic examination of suspension- cultured cells of Phleum pratense L., Panicum miliaceum L., Phalarisaquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undifferentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%).

Cell Wall↗
PubMed1998

3-(Iodoacetamido)-benzoylurea: a novel cancericidal tubulin ligand that inhibits microtubule polymerization, phosphorylates bcl-2, and induces apoptosis in tumor cells.

3-(Iodoacetamido)-benzoylurea (3-IAABU) is a newly synthesized antitubulin compound with a molecular weight of 347. 3-IAABU exhibited anticancer activity in a variety of tumor cell lines with ID90 in the range of 0.015-0.29 microM for leukemic cells and 0.06-0.92 microM for solid tumors. Higher selectivity against malignant cells was observed with 3-IAABU than that with vinblastine and paclitaxel. It inhibits microtubule assembly in tubulin systems either with or without microtubule-associated proteins (ID50 was 0.1 microM and 1.2 microM, respectively) and microtubule depolymerization was not affected, indicating an inhibition of polymerization by binding of 3-IAABU to the heterodimeric subunit of tubulin. 3-IAABU was shown to inhibit the binding of colchicine, a subunit binding compound, but did not inhibit binding of vinblastine and guanosine 5'-triphosphate/guanosine 5'-diphosphate, indicating that colchicine site corresponds to the site that 3-IAABU locates. Tumor cells treated with 3-IAABU showed scattered chromosomes in metaphase. Normal microtubule architecture or spindle apparatus was absent in these cells; instead, punctuated aggregates of tubulin were found by an immunofluorescent staining. Cell cycle analyses showed an accumulation of tumor cells at M phase after a 4-h treatment with 3-IAABU. The phosphorylated bcl-2 representative of an inactivated form of the oncoprotein was found in the cells 12 h after treatment with 3-IAABU. These cells progressed to apoptosis within 16 h. As a new tubulin ligand, 3-IAABU could be a promising agent in cancer chemotherapy.

Antineoplastic Agents↗
PubMed1998

Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition.

Limited stability impedes the development of industrial and pharmaceutical proteins. Dried formulations are theoretically more stable, but the drying process itself causes structural damage leading to loss of activity after rehydration. Lyophilization is the most common method used to dry proteins, but involves freezing and dehydration, which are both damaging to protein. We compared an air-drying method to freeze-drying to test the hypothesis that terminal dehydration is the critical stress leading to loss of activity. The secondary structure of air-dried and freeze-dried actin was analyzed by infrared spectroscopy and related to the level of activity recovered from the rehydrated samples. Actin dried by either method in the absence of stabilizers was highly unfolded and the capacity to polymerize was lost upon rehydration. The degree of unfolding was reduced by air-drying or freeze-drying actin with sucrose, and the level of activity recovered upon rehydration increased. The addition of dextran to sucrose improved the recovery of activity from freeze-dried, but not air-dried samples. Dextran alone failed to protect the structure and function of actin dried by either method, indicating that proteins are not protected from dehydration-induced damage by formation of a glassy matrix. In some cases, recovered activity did not correlate directly with the level of structural protection conferred by a particular additive. This result suggests that secondary structural protection during drying is a necessary but not sufficient condition for the recovery of activity from a dried protein after rehydration.

Actins↗
PubMed1994

KAR3-encoded kinesin is a minus-end-directed motor that functions with centromere binding proteins (CBF3) on an in vitro yeast kinetochore.

We have used in vitro motility assays to investigate the mechanism of kinetochore function in the budding yeast Saccharomyces cerevisiae. Functional centromeric DNA plus a tripartite centromere binding protein complex, CBF3, was found to be necessary but not sufficient for in vitro kinetochore activity. A fourth required component was identified as the motor protein Kar3p, a previously reported yeast kinesin known to be involved in karyogamy and mitosis. Our data support genetic evidence suggesting that Kar3p is a kinetochore-associated motor and imply that CBF3 plays a regulatory role in kinetochore function.

Centromere↗
PubMed1993

An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules.

Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B, CBF5p, obtained by overexpression in bacterial cells, binds microtubules in vitro, whereas C-terminal deleted proteins lacking the (KKD/E)n domain do not. Dividing yeast cells containing a C-terminal truncated CBF5 gene, producing CBF5p containing only three copies of the KKD/E repeat, delay with replicated genomes at the G2/M phase of the cell cycle, while depletion of CBF5p arrests most cells in G1/S. Overproduction of CBF5p in S. cerevisiae complements a temperature sensitivity mutation in the gene (CBF2) specifying the 110-kDa subunit of the high-affinity CEN DNA-binding factor CBF3, suggesting in vivo interaction of CBF5p and CBF3. A second low-affinity centromere-binding factor has been identified as topoisomerase II.

Base Sequence↗
PubMed1992

Microtubule-motor activity of a yeast centromere-binding protein complex.

During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.

Adenosine Triphosphate↗
PubMed1991

In vitro studies of water activity and bacterial growth inhibition of sucrose-polyethylene glycol 400-hydrogen peroxide and xylose-polyethylene glycol 400-hydrogen peroxide pastes used to treat infected wounds.

Water activity and bacterial growth inhibition have been studied in formulations comprising either sucrose or xylose along with polyethylene glycol 400 and hydrogen peroxide. The pastes are chemically stable for 6 months if stored at 2 to 8 degrees C and have been shown to lower water activity to levels below those essential for bacterial growth and to be bactericidal even when diluted up to 50% with serum. Of the organisms tested, Staphylococcus aureus proved the least susceptible to the bactericidal effects of these pastes, and candida and gram-negative organisms proved the most susceptible. Pastes without hydrogen peroxide were less rapidly bactericidal than pastes with hydrogen peroxide, while polyethylene glycol 400 itself was found to have considerable antimicrobial activity. It is suggested that sucrose paste may be of benefit as a treatment for infected and malodorous wounds.

Animals↗
PubMed1989

An autopsy-based study of diagnostic errors in geriatric and nongeriatric adult patients.

One hundred sixty-two consecutive adult autopsies (87 of subjects over age 65 years and 75 of subjects aged 23 to 64 years) performed at a university hospital were studied retrospectively by six internists to determine (1) if diagnostic errors were quantitatively or qualitatively different between the two age groups; (2) if the underlying causes of error (divided into nine categories) were different or age related in any way between the two groups; and (3) any aspects of care that related age to clinical outcome. We found the frequency of major clinical/autopsy discrepancies to be similar to those in previous studies (35%), but in only 7% of cases were these likely to have affected therapy/outcome. Ther was no difference in the frequency of major discrepancies between age groups. There were significantly more "unexpected" minor discrepancies in the older patients, probably related to the multiplicity and complexity of their problems, but these would have affected therapy/outcome in only 1 (3%) of 37 cases. The most common causes of 136 clinical "errors" in 151 autopsies were, in order of frequency: a diagnostic "blind spot," a conscious decision not to pursue a clinical finding (not a real "error"), failure to account for a symptom or sign, atypical presentations, and inadequate follow-up of abnormal laboratory findings. There were no differences between the geriatric and adult groups in terms of frequency or cause of the errors. We conclude that (1) there is no difference in the diagnostic accuracy regarding cause of death between geriatric and nongeriatric patients in the acute hospital environment, and (2) closer attention to basic knowledge and clinical skills and a special focus on judgment and reasoning skills, utilizing autopsy findings among other things, will lead to even further improvement in clinical care at all ages.

Adult↗
PubMed1988

Evidence for extracellular deamination of adenosine in the rat heart.

1. In rat heart perfused with adenosine (10(-6) M), dilazep (10(-4) M) inhibited incorporation of adenosine into nucleotides (an index of nucleoside transport and phosphorylation) to a greater extent (70%) than metabolism to inosine and uric acid (40%) and actually increased the recovery of inosine to 30% of the adenosine infused. 2. Extrapolating for complete inhibition of transport suggested that 60% of adenosine metabolism was intracellular and 40% extracellular. 3. Static incubations of atria also gave an estimate for extracellular metabolism of 40%. 4. Adenosine deaminase was localised by immunocytochemistry to the extracellular surface of endothelial cells of small coronary arteries. 5. Extracellular deamination may explain the lack of effect of nucleoside transport inhibitors on responses to adenosine in rat heart.

Adenosine↗
PubMed1984

Effects of myocardial catecholamine depletion on cellular electrophysiology and arrhythmias during ischaemia and reperfusion.

The effect of myocardial catecholamine depletion on cellular electrophysiology and arrhythmias was assessed in Langendorff perfused guinea pig hearts during ischaemia and reperfusion. Myocardial noradrenaline was reduced to 0.17 +/- 0.04 microgram X g-1 by intracardiac injection of 6-hydroxydopamine (450 mg X kg-1 in six doses over 20 days) compared with 1.5 +/- 0.2 microgram X g-1 in vehicle injected controls. Myocardial catecholamine depletion significantly reduced the incidence of ventricular tachycardia and fibrillation during 30 min of global ischaemia and subsequent reperfusion. Myocardial catecholamine depletion prolonged action potential duration and refractory period during control perfusion and blunted ischaemia induced reduction in action potential amplitude, Vmax, and duration, but accentuated the prolongation in conduction time and QRS width. Catecholamine depletion abolished or attenuated reperfusion induced shortening of action potential duration and refractory period. Catecholamine depletion increased myocardial glycogen levels from 2.47 +/- 0.3 mg X g-1 wet weight to 4.39 +/- 0.3 mg X g-1; fasting animals for 48 h prior to study reversed this with no attenuation of the electrophysiological or antiarrhythmic action. These results provide further evidence that release of endogenous myocardial catecholamines contributes to the electrophysiological changes and arrhythmias associated with myocardial ischaemia and reperfusion.

Action Potentials↗