Different molecular forms of uncomplexed prostate specific antigen (PSA) show similar immunoreactivities.

PSA exists in multiple molecular forms in serum, with the majority complexed to proteinase inhibitors such as alpha 1-antichymotrypsin and alpha 2-macroglobulin. The uncomplexed, or "free" forms of PSA represent a very heterogenous distribution of molecular isoforms. It has been suggested that these variations in uncomplexed PSA may cause differences in their immunologic characteristics which may lead to analytical differences between various PSA assays. We report that various isoforms of uncomplexed PSA purified from seminal fluid as previously described show no differences in relative immunoreactivity and demonstrate equimolar behavior as measured by the TOSOH AIA-600 assay, which is a PSA assay based upon monoclonal PSA and monoclonal detecting antibodies (mono-mono). Furthermore, we show that carbohydrate side-chain modification does not change the equimolar immunoreactivity of these isoforms.

Evolution of web site design: implications for medical education on the Internet.

Since its inception, the world wide web (WWW) has possessed the potential for becoming a 'watershed' medium for conveying complex, structured information across vast temporal and geographical barriers. In 1995, the MedWorld project (http:(/)/medworld.stanford.edu) was created at the Stanford University School of Medicine in an effort to innovate and explore the design process of creating WWW applications specifically for medical education. Until recently, the evolution of WWW applications has been mainly driven by technological advances in client-server technology, enabling or translating traditional modes of collaborative medical education (e.g. voice, presence, print, motion) into WWW devices and applications. Many of these applications, while technologically advanced, lack focused development of interface and interactivity design, which may enhance learning experiences. WWW applications which incorporate design innovation in parity with advances in client-server technology have been termed, 'third generation' web sites and have the potential to improve the quality of WWW applications designed for medical education. This work describes how the MedWorld project has created a 'third generation' WWW application by utilizing innovation in information, interface and interactivity design to create innovative WWW technology for the medical education arena.

One-step HPLC purification procedure for porcine brain 90-kDa heat shock protein.

The 90-kDa heat shock protein (HSP90) was purified from porcine brain by a novel single-step purification procedure using diethylaminoethyl high-performance liquid chromatography (HPLC). About 4.8 mg of HSP90 was isolated from 25 g wet wt porcine brain tissue. The purified protein possessed a single moiety on one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. Western blotting using monoclonal antibody prepared against human HSP90 confirmed its identity as HSP90. These results indicate that small-scale HPLC purification of HSP90 from porcine brain tissue can be readily accomplished, with high yield, using a convenient one-step purification method. The procedure described in this paper represents a significant improvement in current purification methods for the isolation of HSP90 from porcine brain.

Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface.

Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.