Structures of Thymosin Proteins.

The thymosin proteins are all short, highly charged, intrinsically unstructured proteins under natural conditions. However, structure can be induced in many of the thymosin proteins by providing charge neutralization at low pH or by the addition of Zn(2+) ions, organic reagents such as trifluoroethanol, hexafluoropropanol, or n-dodecyltrimethylammonium bromide, or interactions with their natural binding partner proteins. The differing structures of thymosin alpha and thymosin beta proteins have been studied by circular dichroism, nuclear magnetic resonance, and crystallographic methods in order to better understand the role of these proteins. In this structural biology review the structures of prothymosin, parathymosin, thymosin alpha-1, and several beta thymosin proteins, in both native states and under secondary structure-inducing conditions are discussed.

Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein expressed in yeast.

PIXY321, a human cytokine analog genetically engineered by the fusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), was expressed in yeast under the control of the alcohol dehydrogenase 2 (ADH2) promoter and the alpha-mating factor expression system. To provide the material necessary for the evaluation of PIXY321 in clinical trials, the production was scaled up to the 1200-1 scale and the PIXY321 molecule isolated by four successive steps of ion-exchange chromatography. Multiple heterogeneities, due to the presence of different patterns of glycosylation as well as multiple amino acid sequences at both N and C termini, were characterized on the purified molecule using complementary analytical techniques including electrophoresis, liquid chromatography and electrospray mass spectrometry. Four different N-terminal sequences were identified but simplified to a reproducible ratio of two sequences, the mature form and a form starting at Ala3, by adjustment of the process conditions. Molecules lacking 1-6 residues at the C-terminus were identified and their relative frequencies quantified. Amino acid modifications, such as three oxidized Met residues at positions 79, 141 and 187 and one deamidated Asn residue at position 176, were detected at low level. Microheterogeneities in glycosylation were characterized on four different sites, one located in the GM-CSF portion and three in the IL-3 portion of the molecule. The sites were shown to be differentially occupied and to carry 0-10 mannose residues according to their location in the sequence. Precise measurement of the heterogeneities at the molecular level were used to tune the process conditions and ensure reproducibility of the clinical product between lots.

Variability in psychometric test scores: the importance of the practice effect in patient study design.

1. Practice effect was determined for six types of psychometric tests given on eight occasions. 2. No plateau occurred. Stepwise mean decrease in Choice Reaction Time, Short Term Memory Errors, and Card Sorting Time, and stepwise mean increase in Mathematical, Visual Vigilance, and Digit-Symbol Substitution scores developed progressively. 3. Pre-trial training will usually fail to prevent practice effect from confounding effect of any intervention. 4. Design of drug intervention studies must take account of the continuing practice effect.

Effects of methyldopa on psychometric performance.

Double-blind, crossover comparisons of methyldopa with placebo were performed in 16 patients with mild to moderate hypertension. Methyldopa (250 mg tid) for 14 days significantly reduced blood pressure, impaired card-sorting time and digital symbol substitution score, and caused trends for impairment of other psychometric tests. Effects of practice were greater than those of treatment on all tests other than card sorting, although no interaction between treatment and practice was seen. Continued dosage impairs psychometric performance, but practice effect is a major confounder. Simple crossover studies are inadequate for detecting moderate drug effects on intellectual performance.

Placement of small lipovitellin subunits within the vitellogenin precursor in Xenopus laevis.

N-terminal amino acid sequence data for the small lipovitellin subunits in Xenopus laevis crystalline yolk platelets indicate that LV2 beta is derived from vitellogenin A2 and that LV2 tau is most likely derived from vitellogenin A1. The small lipovitellin subunits apparently commence within the exon 24 region of the parental vitellogenins, flanking the C-terminal end of phosvitin. As a consequence, we conclude that most of the vitellogenin sequence encoded by exons 30 to 35 is not accounted for by the known yolk proteins.

The effect of dietary fiber on the bioavailability of digoxin in capsules.

Sixteen healthy volunteers were regularly given 0.4 mg of digoxin daily as two capsules with breakfast. After ten days during which breakfast was supplemented with 11 g of bran fiber, steady-state predose mean serum digoxin was lower (0.89 +/- 0.19 versus 0.84 +/- 0.18 ng/mL, P less than .05) and mean 24-hour area under curve determination was lower (30.5 +/- 6.1 versus 28.4 +/- 6.0 ng X hr/mL, P less than .05) than during the control period without bran. Height and time of peak serum digoxin, and 24-hour urinary digoxin were not significantly different. The 6 to 7% reduction in digoxin absorption from capsules is less than that reported from tablets and is probably clinically unimportant.

The comparative effects of verapamil and a new dihydropyridine calcium channel blocker on digoxin pharmacokinetics.

Conflicting conclusions have been reported about interaction of calcium channel blockers with digoxin. The effects of verapamil (240 mg/day) and a new dihydropyridine calcium channel blocker, isradipine (15 mg/day), on the pharmacokinetics of 1 mg intravenous digoxin were compared. All 24 volunteer subjects were healthy, male, nonobese, and aged 18 to 38 years. Groups of 12 subjects received each oral agent over 15 days, with collections of blood and urine for 72 hours after intravenous digoxin. Significant (P less than 0.05) reduction in nonrenal (7.01 +/- 1.97 to 4.00 +/- 1.86 L/hr) and total clearance (14.1 +/- 2.6 to 11.5 +/- 2.5 L/hr) were induced by verapamil, without change in renal clearance. A near-significant (P less than 0.1) increase in peripheral volume of distribution contributed to prolonged elimination half-life (23.1 +/- 4.4 to 34.3 +/- 9.7 hours). By contrast, isradipine caused only a 9% reduction in volume of distribution. Verapamil causes digoxin accumulation by reducing nonrenal elimination. No evidence of clinically relevant interaction of isradipine with digoxin was seen.

The effect of once-daily minoxidil on blood pressure and plasma lipids.

Once-daily minoxidil administration was added to the treatment regimen of 11 patients with hypertension that was inadequately controlled by nadolol 160 mg and chlorthalidone 50 mg given once daily. Additional diuretic therapy was needed by five patients. During treatment without minoxidil and at three and six months of maintenance minoxidil therapy, respectively, 24-hour postdose supine blood pressure fell significantly (P less than .01) from 142 +/- 19/96 +/- 6 mm Hg to 132 +/- 16/87 +/- 4 and 131 +/- 12/87 +/- 4 mm Hg, and home recordings showed that minoxidil induced a mean increase of about 5 beats/min in heart rate. Resting plasma renin activity was not significantly altered. High-density lipoprotein (HDL) cholesterol increased from 31.6 +/- 9.9 to 35.2 +/- 10.9 mg/dL (P less than .05) and to 34.4 +/- 11.3 mg/dL (NS), and low-density lipoprotein (LDL) cholesterol decreased from 146 +/- 36 to 136 +/- 32 mg/dL (P less than .05) and to 126 +/- 35 mg/dL (P less than .01) over the same periods. At six months, approximately 20% changes in ratios of HDL cholesterol to either LDL or total cholesterol were seen. These changes occurred despite a three-pound mean increase in body weight (P less than .05); these alterations are potentially beneficial in terms of reducing the estimated risk of coronary artery disease.