The Occlusion of Rb(+) in the Na(+)/K(+)-ATPase. I. The identity of occluded states formed by the physiological or the direct routes: occlusion/deocclusion kinetics through the direct route.

Occlusion of K(+) or its congeners in the Na(+)/K(+)-ATPase occurs after K(+)-dependent dephosphorylation (physiological route) or in media lacking ATP and Na(+) (direct route). The effects of P(i) or ATP on the kinetics of deocclusion of the K(+)-congener Rb(+) formed by each of the above mentioned routes was independent of the route of occlusion, which suggests that both routes lead to the same enzyme intermediate. The time course of occlusion via the direct route can be described by the sum of two exponential functions plus a small component of very high velocity. At equilibrium, occluded Rb(+) is a hyperbolic function of free [Rb(+)] suggesting that the direct route results in enzyme states holding either one or two occluded Rb(+). Release of occluded Rb(+) follows the sum of two decreasing exponential functions of time, corresponding to two phases with similar sizes. These phases are not caused by independent physical compartments. The rate constant of one of the phases is reduced up to 30 times by free Rb(+). When Rb(+) is the only pump ligand, the kinetics of occlusion and deocclusion through the direct route are consistent with an ordered-sequential process with additional independent step(s) interposed between the uptake or the release of each occluded Rb(+).     

Collagen I Self-Assembly: Revealing the Developing Structures that Generate Turbidity

Type I collagen gels are routinely used in biophysical studies and bioengineering applications. The structural and mechanical properties of these fibrillar matrices depend on the conditions under which collagen fibrillogenesis proceeds, and developing a fuller understanding of this process will enhance control over gel properties. Turbidity measurements have long been the method of choice for monitoring developing gels, whereas imaging methods are regularly used to visualize fully developed gels. In this study, turbidity and confocal reflectance microscopy (CRM) were simultaneously employed to track collagen fibrillogenesis and reconcile the information reported by the two techniques, with confocal fluorescence microscopy (CFM) used to supplement information about early events in fibrillogenesis. Time-lapse images of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml acid-solubilized collagen I gels forming at 27°C, 32°C, and 37°C were collected. It was found that in situ turbidity measured in a scanning transmittance configuration was interchangeable with traditional turbidity measurements using a spectrophotometer. CRM and CFM were employed to reveal the structures responsible for the turbidity that develops during collagen self-assembly. Information from CRM and transmittance images was collapsed into straightforward single variables; total intensity in CRM images tracked turbidity development closely for all collagen gels investigated, and the two techniques were similarly sensitive to fibril number and dimension. Complementary CRM, CFM, and in situ turbidity measurements revealed that fibril and network formation occurred before substantial turbidity was present, and the majority of increasing turbidity during collagen self-assembly was due to increasing fibril thickness.     

Evaluation of a Commercial Latex Agglutination Test Kit for Cryptococcal Antigen

Two dozen Crypto-LA kits for detecting Cryptococcus neoformans capsular polysaccharide antigens were evaluated. Ten kits proved reliable for detecting and titering antigen in clinical materials. Fourteen kits were found to be inadequate.