The quantitative attributes of human leukocyte proteins detected by silver staining two-dimensional electrophoresis (2-DE) gels were studied by using computer-assisted data analysis. Experiments included (a) analysis of replicate patterns of the same sample, (b) analysis of different dilutions of the same sample, and (c) analysis of samples from different individuals. Over 200 proteins were observed to have coefficients of variation (CV) less than or equal to 15% when data from replicate patterns were analyzed. In contrast, 8 proteins had CV values of less than or equal to 15% when data from different samples were analyzed. The dilution experiment showed that a majority of the proteins detected with some consistency (i.e., observed in at least 80% of the patterns) have a linear relationship between the amount of protein loaded onto a 2-DE gel and the spot volume in the final 2-DE pattern. The slope of the curves and the deviation from linearity were found to be quite protein-specific. These results indicate that optimization of sample purity and minimization of staining protocol variables are required to limit the background quantitative variability between and within 2-DE runs to a level that will allow detection of quantitative changes indicative of biological responses.

Blood Platelets↗

PubMedDate not supplied

Large-scale oligonucleotide synthesizers. I. Basic principles and system design.

The central problem in scaling up oligonucleotide synthesis is to expose each element of a large bed to the same conditions obtaining in very small ones, for the same intervals of time. Our analysis suggests that scale-up is chiefly limited by fluid path length through the bed. By using annular beds in zonal centrifuge rotors of unique design, with fluid flow controlled by combining centrifugal force with differences in physical density between reagents, reagent fronts may be kept exactly perpendicular to the direction of flow in each bed element. Under these conditions, bed volume may be increased by increasing rotor length and diameter. The rotor is lined with polypropylene or Teflon, and has a thick tempered glass end window. Transparent rotary valves of a unique design allow any of 47 reagents to be selected and the direction of flow through the rotor to be controlled. A photodiode spectrophotometer provides complete absorption spectra on fluid in the rotor inlet and outlet lines every 4 s, and a large balance weighs effluent from the synthesizer continuously. The entire operation is controlled by a work station, and steps may be programmed by time, absorbance, or reagent mass. Reagents are identified by spectra, and trityls are integrated on line. A detailed time-stamped log file provides a complete record of each synthesis.

Equipment Design↗

PubMedDate not supplied

Metabolic stability and tumor inhibition of bombesin/GRP receptor antagonists.

Small cell lung cancers (SCLC) synthesize and secrete bombesin/gastrin releasing peptide (BN/GRP). The autocrine growth cycle of BN/GRP in SCLC can be disrupted by BN/GRP receptor antagonists such as [Psi13,14]BN. Here several BN analogues were solid-phase synthesized and incubated with intact SCLC cells at 37 degrees C in RPMI medium in a time-course fashion (0-1080 minutes) to determine enzymatic stability. The proteolytic stability of the compounds was determined by subsequent HPLC analysis. The metabolic half-life ranged from 154 minutes to 1388 minutes for the six analogues studied. [Psi13,14]BN was found to be very stable to metabolic enzymes (T1/2 = 646 mm) and also inhibited SCLC xenograft formation in vivo in a dose-dependent manner. When [Psi13,14]BN was incubated with NCI-H345 cells, it inhibited 125I-GRP binding with an IC50 value of 30 nM. These data suggest that BN/GRP receptor antagonists such as [Psi13,14]BN may be useful for the treatment of SCLC.

Amino Acid Sequence↗