Porcine spleen deoxyribonuclease II. Covalent structure, cDNA sequence, molecular cloning, and gene expression

Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked alpha.beta heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708-10713). The alpha subunit, after disulfide cleavage, yields two chains, alpha1 and alpha2. The complete amino acid sequences of the alpha1, beta, and alpha2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between alpha1 and alpha2 and of three intrachain disulfides in alpha2 were assigned. Six carbohydrate attachment sites, two in beta and four in alpha2, were detected by sugar analyses. The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined. The amplified fragments shown to be a cDNA sequence of 1,292 bases. This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH2-end, two small connecting peptides in the middle, and a peptide at the COOH terminus. These are evidently removed to form mature DNase II. Thus, all three chains in the sequence alpha1, beta, and alpha2 are coded by the same cDNA. When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.     

Knowledge of New English vocabulary in amnesia: an examination of premorbidly acquired semantic memory

To assess if amnesics have intact remote memory for general semantic information, we examined memory for vocabulary words with known dates of entry into the language between 1955 and 1989. Amnesics of mixed etiology with acute onset performed normally on both a recall and a recognition task. Korsakoff patients, in contrast, were impaired on both tasks and demonstrated a gradient such that their knowledge of words acquired during more recent time periods was worse than that of words acquired during more remote time periods. The improvement in performance associated with recognition testing was larger for Korsakoff patients than for control subjects and correlated significantly with a composite measure of frontal dysfunction. These findings suggest a deficit in the controlled search and retrieval of semantic information in Korsakoff patients. The implications of the differential performance of Korsakoff and mixed etiology amnesics for explanations of temporally graded retrograde amnesia are discussed.     

Analytical performance of the Tandem-R free PSA immunoassay measuring free prostate-specific antigen

The analytical performance of the Tandem-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA-alpha 1-antichymotrypsin was determined to be < 1%. The minimum-detectable concentration was < 0.05 microgram/L. The within-run and between-day CVs were < or = 5% for samples with > 0.3 microgram/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.     

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.     

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).