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Four novel proteins (phoratoxins C-F) have been isolated from the North American mistletoe Phoradendron tomentosum. The amino acid sequences of these phoratoxins were determined unambiguously using a combination of Edman degradation and trypsin enzymatic digestion, and by electrospray ionization tandem mass spectrometry sequencing. Phoratoxins C, E and F consist of 46 amino acid residues; and phoratoxin D of 41. All proteins had six cysteines, similar to the earlier described phoratoxins A and B, which are thionins. The cytotoxicity of each protein was evaluated in a human cell line panel that represented several cytotoxic drug-resistance mechanisms. For the half-maximal inhibitory concentrations (IC50 values) of the different cell lines in the panel, correlation with those of standard drugs was low. The most potent cytotoxic phoratoxin C was further tested on primary cultures of human tumor cells from patients. The solid tumor samples from breast cancer cells were 18 times more sensitive to phoratoxin C than the tested hematological tumor samples.

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The goal of the present study was to measure the levels of DNA adducts in human nasal mucosa cells and in total white blood cells in relation to smoking. DNA was isolated from samples of 20 healthy volunteers (six smokers and 14 non-smokers). The levels of DNA adducts were measured by 32P-postlabelling assay. In smokers the mean DNA adduct levels were 3.3 and 17.0 adducts/10(8) nucleotides in total white blood cells and nasal mucosa cells respectively. The corresponding values in non-smokers were 2.0 and 6.8 adducts/10(8) nucleotides. The mean adduct level was significantly higher in nasal mucosa cells than in total white blood cells both in smokers and non-smokers. The mean adduct levels in smokers' nasal mucosa cells were significantly higher than those in non-smokers. Thus the nasal mucosa cells constituted a sensitive tissue for the determination of cigarette smoking induced DNA adducts. Combining the sensitivity of the 32P-postlabelling assay with the specificity of the nasal mucosa to the airborne chemical exposures, the DNA adduct analysis from human nasal mucosa cells represents a method of choice in the assessment of exposure to airborne carcinogens.

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Human exposure to chemical compounds, often termed xenobiotics, has been linked to a number of enhanced incidences of various neoplasias. A majority of these enter the human body through inhalation. Most xenobiotics are metabolized in the body to more hydrophilic metabolites before excretion in the urine and bile. During this process, promutagens and procarcinogens could be activated and could interact with proteins as well as DNA to form adducts. DNA adducts formed by chemical carcinogens can, therefore, be used as biomarkers of exposure and other host factors. This study that DNA adduct analysis can be performed on cells from human nasal mucosa. Using the nasal lavage procedure performed on 20 healthy volunteers, 5 x 10(5) to 5 x 10(6) cells were obtained from which 5 to 40 micrograms DNA was isolated. DNA adducts were analyzed by the 32-P-postlabeling assay. The DNA adduct levels ranged between 1.4 and 6 adducts/10(8) nucleotides. In addition to its simplicity, the nasal lavage procedure is an inexpensive, noninvasive procedure that requires no anesthetics or special equipment. Moreover, the cells obtained are the first to come in contact with air pollutants. DNA adduct analysis from human nose mucosa cells could therefore be used to develop a technique suitable for the assessment of exposure to chemical carcinogens through inhalation.

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The pharmacokinetics of a single oral dose of 1.75 mg glibenclamide were studied in 15 healthy Caucasians including five poor metabolisers of debrisoquine and five poor metabolisers of S-mephenytoin. Plasma glibenclamide concentrations and the urinary concentrations of trans-4- and cis-3-hydroxyglibenclamide were analyzed by h.p.l.c. Thirty-six +/- 6% (mean +/- s.d., n = 15) of the given dose of glibenclamide was excreted in 48 h urine as hydroxylated metabolites, 27 +/- 4% as trans-4-hydroxyglibenclamide and 8 +/- 2% as cis-3-hydroxyglibenclamide. There were no differences in the plasma pharmacokinetics of glibenclamide or in the urinary excretion of the metabolites between poor and extensive metabolisers of debrisoquine, neither between the two mephenytoin hydroxylator phenotypes. The study thus indicates that the disposition of glibenclamide is not influenced by these two independent polymorphisms of drug oxidation.

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The effect of glipizide on hepatic uptake of insulin was studied in five patients with liver cirrhosis and five patients with varying diseases in the biliary system and pancreas. All patients had portal catheters for diagnostic purposes. The hepatic uptake of insulin was estimated from the clearance rate for insulin obtained after a constant rate infusion into a peripheral vein and the portal vein. Each patient was examined on two consecutive mornings, the second investigation carried out one hour after oral glipizide administration. The fractional hepatic uptake was significantly lower in cirrhotic patients (17% +/- 6%) than in the other patients (52% +/- 7%; P less than .01). After glipizide, an increase in the estimated uptake of insulin occurred in cirrhotic patients (from 17% +/- 6% to 39% +/- 6%; P less than .01), whereas an insignificant decrease was observed in the other patients (from 52% +/- 7% to 43% +/- 11%).

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Eight patients aged 57-86 years with non-insulin-dependent diabetes mellitus (NIDDM) taking long-term glibenclamide treatment (5-15 mg/day) were given oral trimethoprim-sulphamethoxazole due to an acute bacterial infection. After 4-6 days of combined treatment, the total plasma glibenclamide concentrations were determined every second hour for 12 h, and the area under the curve (AUC) was computed. A second plasma glibenclamide profile was obtained 2-4 weeks after withdrawal of trimethoprim-sulphamethoxazole. The levels of plasma glibenclamide did not differ between the two occasions, nor did the simultaneously determined levels of blood glucose and plasma insulin. It is concluded that there is no consistent pharmacokinetic interaction between glibenclamide and trimethoprim-sulphamethoxazole in NIDDM patients.

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The mistletoe protein toxins ligatoxin, phoratoxins A and B, and viscotoxins A3 and B have been investigated by 1H NMR spectroscopy at 300 and 600 MHz. The five polypeptides define a set of closely related homologues, containing 46 amino acid residues each, in a structure constrained by three cystine bridges. Their methyl and aromatic spectra were analyzed and a number of signals identified and assigned via comparative criteria, two-dimensional chemical-shift correlated spectroscopy, acid-base titration, and proton Overhauser experiments in 1H2O. The spectra indicate a compact globular conformation and a common folding pattern for the toxins. In particular, use was made of well-resolved aliphatic and aromatic resonances in order to compare the mistletoe proteins with the thionins, a set of homologous toxins from gramineae, and with crambin, a closely related polypeptide from a crucifer, which we have previously studied by NMR. We observe that while all the investigated proteins have very similar secondary and tertiary structures, they differ widely in their dynamic characteristics as probed by the amide NH 1H-2H exchange kinetics in deuteriated solvents; thus, while crambin and the thionins exhibit very fast isotope exchange, the kinetics for the mistletoe toxins are slow, with some NH groups showing exchange half-lives that extend up to several days at pH* 5.8 or that are too long to be measurable at ambient temperature. The temperature dependence of the 1H NMR spectrum also indicates that the toxins are endowed with a thermally very stable native (ground-state) structure, with little evidence of large amplitude structural breathings up to approximately 370 K, although irreversible chemical degradation (denaturation) becomes evident at temperatures greater than or equal to 350 K. It is concluded that the mistletoe toxins afford valuable rigid structures for NMR conformational studies.

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