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Biomedical subjects

A Cooper

Publications and source records attributed to A Cooper.

At least 163 records · Page 9Linked to original sources
PubMed1996

Direct measurement of the association of a protein with a family of membrane receptors.

A specific receptor is a requirement for most protein toxins and OmpF, a trimeric porin, was previously considered to be the unique membrane-receptor for colicin N. We show by qualitative in vivo analysis that the related porins OmpC or PhoE act as much less effective receptors. To elucidate receptor function, the in vitro binding of the 42 kDa toxin to each of the 120 kDa porin trimers was determined quantitatively using isothermal titration calorimetry. Colicin N binds to OmpF with Ka approximately 5 x 10(5) M-1 and a stoichiometry consistent with about three per trimer but it also binds to PhoE and OmpC with surprisingly similar affinities and stoichiometry. However, thermodynamic analysis of these hitherto unmeasured interactions suggests an unexpected entropic difference between these protein import receptors.

Bacterial Outer Membrane Proteins↗
PubMed1996

Energetics of cyclodextrin-induced dissociation of insulin.

The energetics of dissociation of bovine insulin in aqueous solution have been investigated by sensitive dilution microcalorimetry. Cyclodextrins increase dissociation of insulin oligomers in a manner consistent with their interaction with protein side chains. For example, assuming monomer-dimer equilibrium, in the absence of cyclodextrins the calorimetric dilution data (25 degrees C, pH 2.5) indicate a dimer dissociation constant (K(diss)) of about 12 microM and an endothermic dissociation enthalpy (delta H(diss) of +41 kJ mol-1. Addition of methyl-beta-cyclodextrin (up to 200 mM) makes dissociation significantly more endothermic (delta H(diss) = 79 kJ mol-1) and reduces the apparent dimer dissociation constant by more than two orders of magnitude (K(diss) approximately 1.7 mM). Qualitatively similar results are observed with alpha-cyclodextrin and other beta-cyclodextrin derivatives. Cyclodextrin-induced insulin dissociation is also observed at pH 7.4.

Animals↗
PubMed1996

Mucopolysaccharidosis IVA: a comparative study of polymorphic DNA haplotypes in the Caucasian and Japanese populations.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulphate sulphatase (GALNS). The genetic heterogeneity at the GALNS locus was studied in 62 mutant alleles and 376 normal alleles in the Caucasian population and also in 40 mutant and 100 normal alleles in the Japanese population. For this study, six different restriction fragment length polymorphisms (RFLPs) at the GALNS locus were analysed to search for the frequency of each RFLP produced by StyI, SphI, RsaI, HaeIII, StuI and HapII restriction endonucleases. We detected a total of 27 haplotypes in the Caucasian and Japanese population. Of these 27 haplotypes, 18 haplotypes were present in the Caucasian population and the most common of these was haplotype 1 (ABHcde) in both mutant and normal alleles. In contrast, in the Japanese population we found 20 of the 27 haplotypes and the most common in mutant and normal alleles was haplotype 2 (abhcDE). Within these two populations a parent in the MPS IVA family has an average probability of greater than 77% (in the Caucasian population 77.27% and in the Japanese population 78.26%) of being heterozygous, and hence informative for linkage, at one or more GALNS RFLP sites. Our results delineate the molecular heterogeneity of MPS IVA haplotypes, as well as their significant interpopulation variation, and make prenatal diagnosis and carrier detection possible in the majority of families with one affected child.

Acetylgalactosamine↗
PubMed1996

Blood conservation in small adults undergoing valve surgery.

OBJECTIVES: A substantial reduction in transfusion requirements for cardiac surgical procedures has been reported. Many of these reports have been described in patients undergoing coronary artery bypass grafting. Patients suffering from rheumatic heart disease in India are usually small and also anemic. This study was conducted to assess blood conservation methods for cardiac valve surgery in this subset of patients. DESIGN: This was a prospective, randomized study. SETTING: The study was performed in a New Delhi tertiary care hospital, and the patients were referred from the northern states of India. PARTICIPANTS: One hundred fifty consecutive patients undergoing elective valve surgery using cardiopulmonary bypass were included. The mean age was 27.7 years and mean weight was 45.2 kg. INTERVENTIONS: The patients were divided into three groups of 50 each. Group 1 received autologous fresh blood donated before bypass, and both a cell saver and membrane oxygenator were used. The oxygenator contents at the end of perfusion were processed by cell saver. Group 2 patients were reinfused with autologous blood only, and group 3 was a control group. In groups 2 and 3, the blood that remained in the oxygenator at the conclusion of cardiopulmonary bypass was reinfused. A hematocrit of less than 25% was considered an indication for transfusion in the postoperative period. MEASUREMENTS AND MAIN RESULTS: The mean preoperative hematocrit was 35.5%. A mean of 361.1 mL of autologous blood was collected from group 1 and 303.3 mL from group 2. Group 1 required 15 units of bank blood, group 2, 90 units (p < 0.001), and group 3, 102 units (p < 0.001). Seventy-eight percent of group 1 patients did not receive any donor blood. There was no significant difference in chest tube drainage among the three groups. CONCLUSIONS: In this unique group of patients whose mean body weight was only 45 kg, autologous blood alone did not decrease blood bank requirements but when combined with a cell saver and membrane oxygenator greatly reduced the need for donor blood.

Adult↗
PubMed1996

Confocal microscopy of idarubicin localisation in sensitive and multidrug-resistant bladder cancer cell lines.

Idarubicin is a highly lipophilic anthracycline and appears effective against tumours resistant to conventional anthracyclines. Confocal microscopy demonstrates predominantly cytoplasmic idarubicin accumulation. This distribution is unaltered by resistance status or the resistance reversing agent verapamil. Our results contrast with studies on conventional anthracyclines and suggest that nuclear accumulation may not be a prerequisite for anthracycline cytotoxicity.

Antibiotics, Antineoplastic↗
PubMed1996

Determination and reversal of resistance to epirubicin intravesical chemotherapy. A flow cytometric model.

OBJECTIVE: To develop a method of determining the characteristics of epirubicin resistance and to study the reversal of such resistance in the intravesical treatment of superficial bladder cancer, using sensitive and resistant derivatives of a bladder cancer cell line in vitro. MATERIALS AND METHODS: Epirubicin fluorescence and flow cytometry were used to measure the intracellular levels of epirubicin in both sensitive and resistant live cultured bladder tumour cells, with and without different doses of the resistance-reversing agent verapamil. RESULTS: There was a reliable, highly significant and consistent difference in intracellular epirubicin concentration between the resistant and sensitive bladder tumour cells. In addition, it was possible to substantially reverse the features of resistant cell subline with additional verapamil. CONCLUSION: Application of this assay to clinical specimens should allow better targeting of epirubicin intravesical chemotherapy and avoid the premature termination of such treatment in patients whose tumours remain sensitive to this agent. Furthermore, the addition of verapamil to intravesical epirubicin may permit effective treatment of those patients whose tumours have inherent or acquired resistance to epirubicin.

Administration, Intravesical↗
PubMed1996

Determination and reversal of resistance to epirubicin intravesical chemotherapy. A confocal imaging study.

OBJECTIVE: To evaluate the use of confocal microscopy in the study of resistance to epirubicin and to determine the effect of temperature, viability and a resistance-reversing agent on the intracellular distribution of this drug in sensitive and resistant derivatives of a superficial bladder cancer cell line. MATERIALS AND METHODS: Viable and non-viable adherent cells were incubated in epirubicin solutions under various conditions. After incubation, the distribution of intracellular epirubicin fluorescence was visualized using confocal microscopy and a x50 water-immersion lens. RESULTS: There was a striking and consistent difference between resistant and sensitive cells in the intracellular distribution of the drug. In addition to having greater overall levels of epirubicin fluorescence, sensitive cells accumulated epirubicin predominantly in the nucleus. Epirubicin fluorescence in resistant cells was cytoplasmic and granular in appearance. When incubated at 0 degrees C, both cell lines showed no nuclear uptake and thus resembled resistant cells at 37 degrees C. However, dead cells rapidly acquired brightly fluorescent nuclei. The resistance-reversing agent verapamil appeared to cause reversion of the resistant to the sensitive phenotype. CONCLUSION: Confocal microscopy allows epirubicin-sensitive and resistant cultured tumour cells to be differentiated reliably and provides information about the mechanisms of action of, and resistance to, epirubicin. Applying this technique to clinical specimens should enable patients who have the resistant phenotype to be detected and the efficacy of intravesical resistance-reversing agents to be evaluated in such cases.

Administration, Intravesical↗
PubMed1996

The costs and benefits of asking patients for their opinions about general practice.

BACKGROUND: Patient views are important in the evaluation of the quality of health care. The use of surveys needs to be evaluated to determine their cost-effectiveness and benefits. OBJECTIVES: To determine the costs of conducting patient opinion surveys in general practice and to find out how effective patient surveys are in stimulating changes which are beneficial for patient care. METHOD: Postal questionnaire to all 102 medical audit advisory groups (MAAGs) and 98 family health services authorities (FHSAs) in England and Wales, followed by postal questionnaire to 302 general practices reported to have conducted surveys, sampled by the type or questionnaire used. Numbers of MAAGs and FHSAs reporting surveys in general practice; types of questionnaire used; estimated costs; changes made; and benefits identified were measured. RESULTS: Eighty-five (83%) MAAGs and 75 (77%) FHSAs responded. One hundred and fifty-four (96%) of MAAGs or FHSAs reported survey activity. Types of questionnaire used were 1) designed by the practice, 2) designed by the MAAG or FHSA, possibly in collaboration with a practice, or 3) standard 'off-the-shelf'. One hundred and thirty-three (44%) practices responded. Total costs to a practice of conducting a survey ranged from nothing to over 2200 Pounds. Questionnaires designed by the practice are likely to be more costly than other designs. Some practices had surveys provided free of charge by MAAG or FHSA. Sixty-one per cent of practices said changes had been implemented and a further 22% of practices said changes were planned. The most common change was to appointment systems. Benefits were identified for patients, staff, the practice, the MAAG or FHSA and the NHS. Surveys also brought benefits in relationships and understanding. Only 8.2% of practices felt the costs of surveys outweighed the benefits. CONCLUSIONS: Many practices are surveying patients' opinions. Surveys can be costly but MAAGs and FHSAs can provide expertise and resources. Surveys using any of the types of questionnaire are likely to lead to changes and identifiable benefits. Benefits of surveys are perceived by the majority of practices to outweigh the costs.

Cost-Benefit Analysis↗
PubMed1996

Conserved sequence motifs, alignment, and secondary structure for the third domain of animal 12S rRNA.

Secondary structure models are an important step for aligning sequences, understanding probabilities of nucleotide substitutions, and evaluating the reliability of phylogenetic reconstructions. A set of conserved sequence motifs is derived from comparative sequence analysis of 184 invertebrate and vertebrate taxa (including many taxa from the same genera, families, and orders) with reference to a secondary structure model for domain III of animal mitochondrial small subunit (12S) ribosomal RNA. A template is presented to assist with secondary structure drawing. Our model is similar to previous models but is more specific to mitochondrial DNA, fitting both invertebrate and vertebrate groups, including taxa with markedly different nucleotide compositions. The second half of the domain III sequence can be difficult to align precisely, even when secondary structure information is considered. This is especially true for comparisons of anciently diverged taxa, but well-conserved motifs assist in determining biologically meaningful alignments. Patterns of conservation and variability in both paired and unpaired regions make differential phylogenetic weighting in terms of "stems" and "loops" unsatisfactory. We emphasize looking carefully at the sequence data before and during analyses, and advocate the use of conserved motifs and other secondary structure information for assessing sequencing fidelity.

Animals↗
PubMed1996

Molecular determinants of acute single-cell lysis by human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4-positive lymphocytes is accompanied by acute cytopathic effects, i.e., syncytium formation and single-cell lysis. Syncytium formation involves cell-cell fusion mediated by viral envelope glycoproteins on the surface of infected cells and by CD4 glycoproteins on adjacent cells. The molecular basis for the lysis of single-HIV-1 infected cells is unclear. Here we report that the expression of functional envelope glycoproteins from primary and laboratory-adapted HIV-1 isolates resulted in the lysis of single CD4-positive lymphocytes. As was previously observed in HIV-1 infected cultures, single-cell lysis in this system primarily involved necrosis and was not inhibited by soluble CD4. Binding of the viral envelope glycoproteins to the CD4 glycoprotein facilitated, but was not sufficient for, cytolysis. Importantly, the ability of the HIV-1 envelope glycoproteins to mediate membrane fusion was essential for single-cell killing. By contrast, the long cytoplasmic tail of the gp41 transmembrane envelope glycoprotein was neither necessary nor sufficient for single-cell lysis. These results suggest that intracellular envelope glycoprotein-CD4 interactions initiate autofusion events that disrupt cell membrane integrity, leading to single-cell lysis by HIV-1.

CD4-Positive T-Lymphocytes↗
PubMed1996

Software to determine optimal oligonucleotide sequences based on hybridization simulation data.

In the design of oligonucleotide sequences for targeting DNA or RNA sequences, it can be difficult to identify sequences that will hybridize only to the intended target. The term "sequence-specific" or "sequence-nonspecific" is often used to describe the interactions of an oligonucleotide with a mixture of DNA or RNA. Our new computer program, HYBsimulator (formerly OligoProbe DesignStation), creates a set of candidate oligonucleotides from a target gene. For each of the candidate oligonucleotides, a large sequence database is searched for sequences that will hybridize to the oligonucleotide. This is referred to as computer hybridization simulation (CHS). Using the nearest-neighbor model, the HYBsimulator takes into account mismatches in hybridization and calculates the melting temperature (Tm) or free energy for hybridization to all sequences in a database. The specificity of each oligonucleotide is then quantified by the number of genes that may hybridize and the predicted Tms or free energies of hybridization to those genes. The CHS data are used to select oligonucleotides based on their specificity with respect to a database.

Base Sequence↗
PubMed1996

Mucopolysaccharidosis IVA: four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency.

We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations. V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resulted in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation.

Adolescent↗
PubMed1996

A study of lipid profile before and after coronary artery bypass grafting.

This study was undertaken to establish the variability in the levels of total cholesterol (TC), total triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol before and after coronary artery bypass graft (CABG) surgery, in order to determine postoperatively when an accurate assessment can be made of the lipid status. During a prospective study over 4 months, fasting venous samples were taken pre- and postoperatively on day 5, and in the 4th, 8th and 12th weeks. Ninety-six patients admitted to the cardiothoracic and cardiac wards for CABG were recruited to the study. The mean preoperative levels were: TC 5.94 (+/- 0.1 mmol/l), LDL cholesterol 4.02 (+/- 0.09mmol/l) and HDL cholesterol 1.00 (+/- 0.03mmol/l). These were significantly different (p < 0.01) from the levels on the 5th postoperative day when the mean level of TC was 4.14 (+/- 0.084mmol/l), LDL cholesterol was 2.45 (+/- 0.07mmol/l) and HDL cholesterol was 0.74 (+/- 0.03mmol/l). By the 4th postoperative week, mean TC (5.73 +/- 0.13mmol/l), LDL cholesterol (3.79 +/- 0.14mmol/l) and HDL cholesterol (1.03 +/- 0.04mmol/l) did not vary significantly from the mean preoperative values. There was no significant difference in the mean TG levels pre- and postoperatively. The mean TC, LDL cholesterol and HDL cholesterol rise to preoperative levels by the 4th week after CABG. Thus, an accurate assessment of patients' lipid status can be made from this period. An earlier postoperative assessment may be falsely reassuring.

Adult↗
PubMed1996

Verbal abuse of hospital staff.

Many researchers have investigated verbal abuse against nurses, but few or no investigators have studied nurses and physicians collectively. At our 650-bed community hospital, anecdotal reports of verbal abuse of nurses by physicians led our research team to investigate.

Humans↗
PubMed1995

Vibrational Raman optical activity of alpha-lactalbumin: comparison with lysozyme, and evidence for native tertiary folds in molten globule states.

Proteins in aqueous solution are now accessible to Raman optical activity (ROA) measurements, which provide an incisive new probe of secondary and tertiary structure illustrated here by a study of bovine alpha-lactalbumin. The room-temperature ROA spectrum of native bovine alpha-lactalbumin is similar to that of native hen egg-white lysozyme except for features attributable to differences in the loop regions: in particular, a positive ROA band at approximately 1338 cm-1 assigned to conformationally homogeneous loop structure, possibly with local order corresponding to 3(10)-helix, has more than double the intensity in alpha-lactalbumin compared with lysozyme. This is consistent with the two proteins having similar secondary structure but different local details in the tertiary fold. ROA measurements on alpha-lactalbumin at pH 2.0 over a range of temperatures have provided a new perspective on the molten globule state. Thus at 35 degrees C ROA reveals the presence of some secondary structure but an almost complete loss of the tertiary loop structure; whereas at 2 degrees C the ROA spectrum is almost identical with that of the native protein, which is strong evidence that virtually all of the secondary structure and the tertiary backbone fold persist, albeit within a looser framework associated with increased solvent exposure and change of environment of many of the side-chains as evidenced by an increase in noise and bandwidth of some of the ROA signals together with aromatic fluorescence and near-UV circular dichroism signals characteristic of the molten globule state. Our sample of acid alpha-lactalbumin at 2 degrees C therefore appears to be an archetypal example of Ptitsyn's "native-like" molten globule, having a fixed native-like tertiary fold but with loss of tight packing of the side-chains; whereas at 35 degrees C it is a "disordered" molten globule. At 20 degrees C the acid molten globule appears to retain highly native-like secondary structure but with most of the tertiary fold already lost. A calcium-free sample of alpha-lactalbumin at neutral pH displayed a broad cooperative transition between native and molten globule states at approximately 15 degrees C, with the latter state showing similar but somewhat degraded tertiary loop ROA signatures to the native protein. In both the acid and apo molten globule states the ROA signatures of the secondary structure and the tertiary loops showed a gradual change with temperature.

Calcium↗
PubMed1995

The Oligocene bottleneck and New Zealand biota: genetic record of a past environmental crisis.

Mitochondrial DNA (mtDNA) 12S sequences from the two New Zealand ratite orders, the kiwi and moa, indicate that each group represents a separate colonization event. However, the amount of mtDNA genetic diversity observed within each of the New Zealand groups is similar, and limited, which is not consistent with the ratite phylogenetic tree. We examine the Cenozoic (65-0 million years ago) geological history of New Zealand for possible causes and summarize evidence for land area and climatic changes. A genetic bottleneck is hypothesized and tested, by using molecular data from a second mitochondrial locus (ND6) and a further taxon, the New Zealand acanthisittid wrens. The data support the existence of a widespread genetic bottleneck during the mid-Cenozoic in New Zealand and demonstrate the benefits of synthesizing traditional research with new molecular techniques.

Animals↗