Amplification refractory mutation system linear extension: a novel, gel-free, enzyme-linked immunoassay method for DNA genotyping

A single synthesis cycle of the amplification refractory mutation system (ARMS) was applied to the analysis of K-ras alleles amplified by polymerase chain reaction and immobilized in streptavidin-coated microtiter plates. The ARMS cycle provided the specificity and molecular switch characteristics of a conventional ARMS assay. This allowed linear extension from an allele-specific primer and the incorporation of digoxigenin-labeled deoxyuridine monophosphate from digoxigenin-11-deoxyuridine triphosphate in the presence of the appropriate K-ras allele. Any digoxigenin-labeled deoxyuridine monophosphate substitution was then demonstrated by enzyme-linked immunoassay with colorimetric endpoint. This method is capable of detecting underrepresented acquired mutations, and this has been shown by the unambiguous detection of specific K-ras mutations in cell line DNA/normal human genomic DNA admixtures. The characterization of K-ras mutations in frozen colorectal tumor samples and histologic material is also described.     

Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q and R

Human serum paraoxonase (PON 1) exists in 2 major polymorphic forms (Q and R), which differ in the amino acid at position 191 (glutamine and arginine, respectively). These PON allozymes hydrolyze organophosphates and aromatic esters, and both also protect LDL from copper ion-induced oxidation. We have compared purified serum PONs of both forms and evaluated their effects on LDL oxidation, in respect to their arylesterase/paraoxonase activities. Copper ion-induced LDL oxidation, measured by the production of peroxides and aldehydes after 4 hours of incubation, were reduced up to 61% and 58%, respectively, by PON Q, but only up to 46% and 38%, respectively, by an equivalent concentration of PON R. These phenomena were PON-concentration dependent. Recombinant PON Q and PON R demonstrated similar patterns to that shown for the purified serum allozymes. PON Q and PON R differences in protection of LDL against oxidation were further evaluated in the presence of glutathione peroxidase (GPx). GPx (0.1 U/mL) alone reduced copper ion-induced LDL oxidation by 20% after 4 hours of incubation. The addition of PON R to the above system resulted in an additive inhibitory effect on LDL oxidation, whereas PON Q had no such additive effect. The 2 PON allozymes also differed by their ability to inhibit initiation, as well as propagation, of LDL oxidation. PON Q was more efficient in blocking LDL oxidation if added when oxidation was initiated, whereas PON R was more potent when added 1 hour after the initiation of LDL oxidation. These data suggest that the 2 allozymes act on different substrates. Both PON allozymes were also able to reduce the oxidation of phospholipids and cholesteryl ester. PON Q arylesterase activity was reduced after 4 hours of LDL oxidation by only 28%, whereas the arylesterase activity of PON R was reduced by up to 55%. Inactivation of the calcium-dependent PON arylesterase activity by using the metal chelator EDTA, or by calcium ion removal on a Chelex column, did not alter PON's ability to inhibit LDL oxidation. However, blockage of the PON free sulfhydryl group at position 283 with p-hydroxymercuribenzoate inhibited both its arylesterase activity and its protection of LDL from oxidation. Recombinant PON mutants in which the PON free sulfhydryl group was replaced by either alanine or serine were no longer able to protect against LDL oxidation, even though they retained paraoxonase and arylesterase activities. Overall, these studies demonstrate that PON's arylesterase/paraoxonase activities and the protection against LDL oxidation do not involve the active site on the enzyme in exactly the same way, and PON's ability to protect LDL from oxidation requires the cysteine residue at position 283.     

Making planned and unplanned role transitions

Changes in organizations mean planned and unplanned role transitions for nurse administrators and managers. Keeping abreast of trends engaging in activities that promote professional growth, assessing work habits, maintaining job interviewing skills, and reviewing and updating resumes are essential to prepare for role changes. When unplanned changes occur because one is fired or a position is abolished, one has to organize personal needs, cope with the job loss, look for a job, and assume a new role. The authors discuss strategies that can be used personally or with others to facilitate job change.     

Acute hormone responses to heavy resistance lower and upper extremity exercise in young versus old men

In the final stage of amyotrophic lateral sclerosis (ALS) the majority of patients develop chronic respiratory failure due to respiratory muscle weakness. The interaction between the patient with ALS and the physician should be characterized by continuous communication, especially with respect to the prospect of ventilatory failure and for support. The patient and his family must be informed thoroughly about the natural history and the prognosis of ALS, depending on the individual disease process. Already in the early stage of the disease coping strategies should be discussed so that imminent respiratory emergencies can be handled. If ALS patients are not informed about the acute respiratory insufficiency they run the risk of having to be intubated and mechanically ventilated over a long term. If dyspnea and hypersecretion dominate the final stage of ALS, the therapeutic strategy consists of the administration of morphine, insufflation of oxygen and bronchoscopic suction. Mechanical ventilation should only be initiated in the exceptional case. However, if dyspnea occurs in the early stage of the disease, when there is no bulbar paralysis and peripheral muscle function is intact, then noninvasive mechanical ventilation via mask may improve the quality of life substantially. Nevertheless, invasive mechanical ventilation via a tracheostomy should be avoided.     

Survey of food safety awareness among HIV positive individuals

Though opioids are known to have neuroprotective properties, little information is available on the functional state of opioidergic receptors following focal cerebral ischaemia. The present study investigated the evolution of the Bmax and Kd for [3H]DAMGO, [3H]DADLE, and [3H]U69,593, respectively, for the mu, delta, and kappa opioidergic receptors after permanent focal cerebral ischaemia in mice. While the various Kd were unchanged, mu and delta Bmax values were precociously decreased in frontoparietal cortices, earlier than kappa receptors, reflecting infarct extension with time. The Bmax values for mu and delta receptors were also altered in non-infarcted tissues, such as tissues at risk (e.g., temporal auditory cortex) and exofocal (e.g., contralateral and non-infarcted) cortices. These results suggest that, in non-infarcted areas, the observed changes reflect functional modifications to focal ischaemia.     

A review of the aetiology of squamous cell carcinoma of the conjunctiva

Squamous cell carcinoma of the conjunctiva is a rare tumour with a multifactorial aetiology. There is strong epidemiological evidence that exposure to solar ultraviolet radiation is an important cause and that HIV infection predisposes to its development. The role of other factors, such as human papillomavirus infection, is unclear.