Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

Na,K-ATPase function in alternating electric fields.

Alternating currents affect ion transport processes and ATP splitting through changes in the activation of the membrane Na,K-ATPase. Both processes vary with the frequency, and the effective range includes the environmental 60 Hz. ATP splitting by Na,K-ATPase suspensions decreases for the enzyme under normal conditions, with the maximum effect at 100 Hz. ATP splitting increases when the enzyme activity is lowered to less than half its optimal value by changes in temperature, ouabain concentration, etc. These observations can be explained by the effects of the ionic currents on ion binding at the enzyme activation sites. Such a mechanism could account for the effects of electromagnetic fields on cells, as the transmembrane enzyme can convey the effect of an extracellular signal into the cell via ionic fluxes, and the measured threshold field is within the range of reported biological effects.     

Long-Term Drug Treatment of Obesity in a Private Practice Setting

ATKINSON, RICHARD L, ROY C BLANK, DONALD SCHUMACHER, NIKHIL V DHURANDHAR, DOUGLAS L RITCH. Long-term drug treatment of obesity in a private practice setting. This study evaluated the long-term efficacy and safety of the combination of phentermine and fenfluramine for the treatment of obesity in a private practice setting. A total of 1388 consecutive, qualified patients presenting to a private general internal medicine practice in Charlotte, NC, were enrolled with eligibility criteria including: age 18 years to 60 years, 20% over “desirable” bodyweight or body mass index <27, no serious medical or psychiatric disease, and no contraindications to drug therapy. Patients were instructed in diet, exercise, and behavior modification techniques and received phentermine (15 mg/day to 30 mg/day) and fenfluramine (20 mg/day to 60 mg/day) continuously for over 3 years. Average duration of treatment was 15. 9 months, and average weight loss at the last visit was 11. 6 kg, or 11. 7% of initial bodyweight. For patients completing 1 year of drug treatment, mean weight loss was 16. 5 kg, or 16% of initial weight. Weight loss persisted for 2 years, but partial regain was seen at 3 years. The dropout rates were 18% at 6 months, 39% at 1 year, 68% at 2 years, and 78% at 3 years. At 1 year, blood pressure of hypertensive patients fell from 151/95 mm Hg to 127/78 mm Hg, and serum cholesterol and triglycerides of hyperlipidemic patients fell by 0. 750 mmol/L (29 mg/dL) and 0. 937 mmol/L (83 mg/dL), respectively. Adverse events were modest. We conclude that, in a private practice setting, long-term treatment of obesity with the combination of phentermine, fenfluramine, and a weight maintenance program is generally safe and effective. More research is needed to determine efficacy and safety for longer than 3 years.     

Demonstration of potent, gonadotropin-like biological activity in the serum of rats during midpregnancy

The gonadotropin-like activity (GnLa) of serum from pregnant rats was measured using the rat interstitial cell testosterone (RICT) bioassay. Serum GnLA was elevated on day 9 of pregnancy, peaked at 7.2 μg rat LH-RPl equivalents/ml on day 11 and declined to undetectable levels by day 15. Serum LH, measured by homologous RIA, was consistently low (<20 ng/ml) during pregnancy, except near term.Rat placental lactogen (rPL), which was measured in the same serum samples by rat radioreceptor assay (RRA), reached maximal concentrations on days 12 and 13 of pregnancy.These data suggest the presence in pregnancy serum of a potent-gonadotropin-like hormone, different from pituitary LH, whose origin is unknown. Furthermore, there are discrepancies between the times of appearance of this GnLA and rPL.     

Addition to the article by Iu. E. Dubrova et al. "Influence of heterozygosity on the variation of anthropometric traits in newborns." Determination of intersample differences by covariation and correlation statistics]

Relationships between heterozygosity of 13 polymorphic loci and variation of the set of anthropometric traits were studied in 302 singleton newborns (174 boys, 128 girls). Statistically significant correlation between heterozygosity and all traits were not found. Variances of four traits--body weight, body length, circumference of head and breast measured by first principal component decrease with increase of heterozygosity in boys and girls. At the same time, variance of body proportions (second principal component) increase significantly in boys only. The highest values of correlation between four traits were found in the group of low heterozygous boys. The groups of newborns with different level of heterozygosity are characterized by different combinations of first principal component and the number of minor deviations from development (stigma). It is concluded from the whole set of data that newborn boys with the average level of heterozygosity have the highest level of viability.     

Erythrocyte shape and volume changes caused by an inhibitor of the glucose and anion transporters

Light scattering measurements and scanning electron microscopy show that p-azidobenzylphlorizin (p-AzBPhz) causes changes in the shape and volume of human erythrocytes by at least two, dose-dependent mechanisms: At nominal concentrations above 5 microM, the azide induces cell swelling by either enlarging a pre-existent channel or by creating pores between phase boundaries of the membrane through which salt and water enter, but sucrose remains excluded. However, over the range 0.03 to 0.3 microM, in either isosmotic NaCl or KCl media, when fewer than 1 million molecules of azide are bound per cell, the ligand causes membrane deformations that convert discocytes into cells resembling stage 2 echinocytes. Whereas a cell volume increase of about 10% accompanies these shape changes, (microhematocrit and electronic cell sizing measurements), no net influx of either Na+ or K+ during this stage of swelling was detectable. These cell alterations take place at p-AzBPhz concentrations which concurrently inhibit both chloride and 3-methoxyglucose equilibrium exchange transport. The results may indicate that when the membrane impermeable p-AzBPhz interacts with the anion and/or sugar transporter, some trans-membrane perturbation occurs which alters the cytoskeleton.