Validation of the adaptive scan method in the quest for time-efficient methods of testing auditory processes.

A major barrier to the clinical application of psychophysical testing of central auditory processes is the time required to obtain precise estimates of different listening abilities. In this study, we validate a novel adaptive scan (AS) method of threshold estimation that is designed to adapt on a range of values around threshold rather than on a single threshold value. This method has the advantage of providing the listener with greater familiarity with the stimulus characteristics near threshold while maintaining precise measurement and increasing time-efficiency. Additionally, we explore the time-efficiency of AS through comparison with two more conventional adaptive algorithms and the method of constant stimuli in two common psychophysical tasks: the detection of a gap in noise and the detection of a tone in noise. Seventy undergraduates without hearing complaints were tested using all four methods. The AS method provided similar threshold estimates with similar precision to those from the other adaptive methods and, thus, it is a valid adaptive method of psychophysical testing. We also provide an analysis of the AS method based on precision metrics to propose a shortened version of the algorithm that maximizes the time/precision tradeoff and can achieve similar thresholds to the adaptive methods tested in the validation. This work lays the foundation for using AS across a wide variety of psychophysical assessments and experimental situations where different levels of precision and/or time-efficiency may be required.

Measurement of tumor markers in chronic hemodialysis patients.

Tumor markers are widely used for screening certain tumors, however, their use in chronic hemodialysis (HD) patients in hemodialysis has been a controversial issue. To determine the reliability of the tumor markers, CA 15-3, CA 19-9, CA 125, alpha-fetoprotein and carcinoembryonic antigen (CEA), in chronic HD patients, and the impact of active hepatitis C on the variation of tumor markers values, we studied 30 patients (16 men and 14 women) aged from 40 to 78 years old (mean age: 54 + or - 5 years), on intermittent hemodialysis (with a mean duration of 10.5 years), and clinically free from neoplastic disease. The control group included 30 healthy volunteers. All subjects were of Greek origin and residents of the Korinthos region. The tumor markers were measured once in the control group and before and afterwards the hemodialysis, in the study group. Alpha fetoprotein was within normal limits in all the study patients, CA 125 was slightly increased in one (3.3%) patient, CA 15-3 levels were twice normal in 4 (13%) patients, CA 19-9 levels were twice normal in 5 (16%) patients, and CEA levels were twice normal in 4(13%) patients. More than half (7/13) of anti HCV positive and all Australian antigen positive patients had abnormal serum levels of CA 15-3 and CA 125 after hemodialysis treatment. We conclude that measurement of some tumor markers such as alfa-fetoprotein may be beneficial in HD patients. However, the elevated levels of other markers including CA 15-3 and CA 125 are not specific for neoplasms and related to active hepatitis C.

Synergy between tetA and rpsL provides high-stringency positive and negative selection in bacterial artificial chromosome vectors.

Bacterial artificial chromosome (bacmid) vectors are used to stably propagate large, complex fragments of cloned DNA and are a core technology for functional genomics. The simplest method of analyzing bacmid clones would involve a direct mutagenesis or allele exchange protocol utilizing positive and negative selectable markers. The utility of three different negative selectable markers to function in the context of a bacmid vector was therefore investigated: sacB from Bacillus subtilis, which confers sensitivity to sucrose; tetA from TN10, which confers resistance to tetracycline, osmotic sensitivity, and sensitivity to kanamycin and streptomycin; and rpsL from Escherichia coli, which confers sensitivity to streptomycin. When expressed individually in the context of a bacmid vector, each of these markers confers a similar stringency of negative selection, with plating efficiencies on selective media of 2.3 x 10(-5), 9.4 x 10(-4), and 5.7 x 10(-5), respectively. However coexpression of rpsL and tetA results in a synergistic enhancement of the osmotic, kanamycin, and streptomycin sensitivities, with a stringency of selection of approximately 50- to approximately 1000-fold over that obtained with rpsL or tetA alone and approximately 20-fold more than that obtained using sacB. The combination of rpsL and tetA thus serves as the most efficient positive and negative selectable marker system described to date.

Expression of the tetA(C) tetracycline efflux pump in Escherichia coli confers osmotic sensitivity.

Expression of tetA(C) in Escherichia coli confers resistance to tetracycline as well as sensitivity to nickel and cadmium salts, lipophilic chelating agents, and aminoglycoside antibiotics. In this report we determine that high-level expression of tetA(C) also confers an osmotic sensitivity. The osmotic-sensitive phenotype is distinct from the tetracycline-resistant phenotype and can be localized to a domain contained within the first 98 amino acid residues of the TetA(C) polypeptide.

An enhanced packaging system for helper-dependent herpes simplex virus vectors.

Helper-dependent herpes simplex virus (HSV) vectors (amplicons) show considerable promise to provide for long-term transduced-gene expression in most cell types. The current packaging system of choice for these vectors involves cotransfection with a set of five overlapping cosmids that encode the full HSV type 1 (HSV-1) helper virus genome from which the packaging (pac) elements have been deleted. Although both the helper virus and the HSV amplicon can replicate, only the latter is packaged into infectious viral particles. Since the titers obtained are too low for practical application, an enhanced second-generation packaging system was developed by modifying both the helper virus and the HSV amplicon vector. The helper virus was reverse engineered by using the original five cosmids to generate a single HSV-bacterial artificial chromosome (BAC) clone in Escherichia coli from which the pac elements were deleted to generate a replication-proficient but packaging-defective HSV-1 genome. The HSV amplicon was modified to contain the simian virus 40 origin of replication, which acts as an HSV-independent replicon to provide for the replicative expansion of the vector. The HSV amplicon is packaged into infectious particles by cotransfection with the HSV-BAC helper virus into the 293T cell line, and the resulting cell lysate is free of detectable helper virus contamination. The combination of both modifications to the original packaging system affords an eightfold increase in the packaged-vector yield.

Renal folate absorption and the kidney folate binding protein. I. Urinary clearance studies.

The kidney possesses a high concentration of a folate binding protein (FBP) that resides primarily in the brush-border membrane (BBM) of the proximal tubular cells. To assess the possible involvement of this protein in renal conservation of folate we determined the urinary clearance, in rats, of three forms of folates with sharply different affinities for FBP. After single intravenous injections of 0.1 to 1.0-nmol doses of radioactive folates the urinary clearance based on radioisotope determination was in the sequence: folic acid less than 5-methyltetrahydrofolate (5-CH3 THF) much less than methotrexate. At higher doses the urinary folate clearance was increased and the differences between the three injected forms were narrowed and were no longer noticeable at 100-nmol doses. Under conditions of continuous infusion to attain plasma folate levels of 2.3-5.7 pmol/ml, the urinary clearance based on chromatographic analyses of plasma and urine after correction for plasma folate binding was 0.20 ml/min for folic acid, 0.37 ml/min for 5-CH3 THF, and 1.76 ml/min for methotrexate. These chromatographic analyses have also shown the presence in both plasma and urine of metabolites formed from infused folates. Metabolites found after infusion of folic acid include 5-CH3 THF with a urinary clearance of 0.3 ml/min and an unknown with a urinary clearance of 0.8 ml/min. The latter metabolite appears also to occur in plasma and urine after infusion of 5-CH3 THF. Infusion of methotrexate was associated with the appearance of a metabolite with a urinary clearance of 2.5 ml/min. This sequence of urinary clearance is in inverse order to the affinities of these three forms of folate for the kidney BBM FBP.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the kidney in metabolism of gonadotropins in rats.

The role of the kidney in the metabolic disposal of homologous gonadotropins [renal luteinizing hormone (rLH) and renal follicle-stimulating hormone (rFSH)] was studied in rats. In analogy with other protein hormones, renal mechanisms contributed importantly to their metabolic clearance rates (MCR), which were profoundly and comparably decreased following nephrectomy (by 94 and 78% for rLH and rFSH, respectively). Absolute MCR and renal organ clearance rates of gonadotropins were, however, markedly lower and urinary clearance rates proportionally higher than those of nonglycosylated protein hormones reported previously. Nonetheless, handling of both LH and FSH by the kidney probably involves, in addition to their excretion in the urine, also intrarenal degradation because their urinary clearance rates accounted for at most a third of their respective MCR, considerably less than the striking reduction of MCR seen after acute renal ablation. Moreover, losses of LH immunoreactivity across the renal circulation were over and above those accountable for by urinary excretion alone. Thus, handling of gonadotropins by the kidney differs from that of nonglycoprotein hormones both in magnitude and in that it involves, in addition to intrarenal degradation, also substantial urinary excretion, a pattern that appears to be representative of the way the kidney disposes of glycoprotein hormones in general.