Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embedded sections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes.     

Mu opioid agonists potentiate amphetamine- and cocaine-induced rotational behavior in the rat

Opioids modulate brain dopaminergic function in various experimental paradigms. This study used the rotational model of behavior in rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal pathway to investigate this interaction. Doses of two presynaptically acting dopaminergic drugs, amphetamine and cocaine, were coadministered with several doses of the mu opioid agonist, morphine. Morphine, at 3.0 mg/kg, potentiated rotational behavior induced by each dose of the stimulants. To determine the receptor specificity of the actions of morphine, the mu opioid agonists buprenorphine, fentanyl, levorphanol, meperidine, and methadone, and dextrorphan, the non-opioid isomer of levorphanol, were administered alone and with 1.0 mg/kg amphetamine. Each of these drugs, as well as morphine, produced circling behavior on its own. All of the mu opioid agonists and dextrorphan increased amphetamine-induced turning; the coadministration of dextrorphan, levorphanol, meperidine, methadone and morphine with amphetamine produced turning greater than predicted by simple additivity. To determine whether an opioid receptor was involved in these interactions, the opioid antagonist, naltrexone, was administered before the amphetamine/mu opioid receptor agonist combination. Naltrexone blocked the potentiating effects of morphine, but not those of the other drugs. Moreover, naltrexone alone dose-dependently increased amphetamine-induced rotational behavior. These studies show that some mu opioid receptor agonists can potentiate stimulant-induced rotational behavior and that blockade of opioid receptors can also produce a potentiation. The role of mu opioid receptors in these effects remains unclear.     

mago nashi mediates the posterior follicle cell-to-oocyte signal to organize axis formation in Drosophila

Establishment of the anteroposterior and dorsoventral axes in the Drosophila egg chamber requires reciprocal signaling between the germ line and soma. Upon activation of the Drosophila EGF receptor in the posterior follicle cells, these cells signal back to the oocyte, resulting in a reorganization of the oocyte cytoplasm and anterodorsal migration of the oocyte nucleus. We demonstrate that the gene mago nashi (mago) encodes an evolutionarily conserved protein that must be localized within the posterior pole plasm for germ-plasm assembly and Caenorhabditis elegans mago is a functional homologue of Drosophila mago. In the absence of mago+ function during oogenesis, the anteroposterior and dorsoventral coordinates of the oocyte are not specified and the germ plasm fails to assemble.     

Medicare spending for elderly beneficiaries who need long-term care

To better understand how elderly people with long-term care needs might be affected by Medicare's greater reliance on risk plans, we examine Medicare spending for this population using data from the Medicare Current Beneficiary Survey. Medicare spending for elderly people with functional limitations is substantially greater than for other beneficiaries, but highly variable. Medicare spends more, however, for community residents with moderate to severe functional limitations than for nursing home residents with similar degrees of limitation. These results raise concerns about the incentives of Medicare risk plans in caring for enrollees with long-term care needs.     

Effect of familial hypophosphatemic rickets on dental development: a controlled, longitudinal study

Familial or X-linked hypophosphatemic rickets (XLHR) is the most common type of rickets in developed countries today. While the dental manifestations of rickets are well reported, there is little information regarding its relationship to dental development and other dental anomalies. This investigation studied the rate of dental development and associated dental anomalies in 19 XLHR subjects compared with 38 race-, age-, and sex-matched control children. The results showed that in both XLHR and control children, no significant differences existed in dental age compared with the respective chronological age, indicating that rickets did not affect the rate of dental development. Longitudinal growth curves of seven XLHR and matched control children substantiated that relationships of dental to chronological ages were comparable in both groups. Male XLHR subjects showed significantly increased tendency for dental taurodontism with mean Crown-Body (CB):Root (R) ratio of 1.1 compared with 1.0 in females and 0.8 in controls (P < 0.02). Male XLHR children also showed significantly increased prevalence (50%) of ectopic permanent canines compared with control children (8%, P < 0.01).     

Impact of chronic pain patients'' job perception variables on actual return to work

A structural model is constructed for the integral membrane protein, sensory rhodopsin I (SRI), the phototaxis receptor of the archaeon Halobacterium salinarium. The model is built on the template of the homologous bacteriorhodopsin (BR). The modeling procedure includes sequence alignment, a side chain rotamer search and simulated annealing by restricted molecular dynamics. The structure is in general agreement with previous results from mutagenesis experiments, chromophore substitution and room and cryogenic temperature spectroscopy. In particular, a residue near the beta-ionone ring of the retinylidene chromophore is found to be critical in maintaining the proper isomeric conformation of the chromophore; a layer of residues lying on the cytoplasmic side of the chromophore pocket is found to modulate the restraints around the C13 region of the chromophore, affecting the isomerizations around its 13 = 14 bond that are important to the protein's activity. The restraints in these regions are more stringent in SRI than in BR. The tightened restraints are chiefly due to van der Waals interactions, where the attractive and repulsive components play separable roles. Aromatic residues account for a majority of the restrictive interactions. It is hypothesized that the enhanced barriers due to these restrictions regulate the progress of SRI's photocycle, so that it can couple with the phototaxis reaction chain in the bacterium. A possibility is also suggested that conformational changes of the protein provide the signal recognized by the transducer.