Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

Role of NK Cell Subsets in Organ-Specific Murine Melanoma Metastasis

Tumor metastasis plays a major role in the morbidity and mortality of cancer patients. Among solid tumors that undergo metastasis, there is often a predilection to metastasize to a particular organ with, for example, prostate cancer preferentially metastasizing to bones and colon cancer preferentially metastasizing to the liver. Although many factors are thought to be important in establishing permissiveness for metastasis, the reasons for organ-specific predilection of each tumor are not understood. Using a B16 murine melanoma model, we tested the hypothesis that organ-specific NK cell subsets play a critical role in organ-specific metastasis of this tumor. Melanoma cells, given intravenously, readily colonized the lungs but not the liver. NK cell depletion (either iatrogenically or by using genetically targeted mice) resulted in substantial hepatic metastasis. Analysis of NK cell subsets, defined by the differential expression of a combination of CD27 and CD11b, indicated a significant difference in the distribution of NK cell subsets in the lung and liver with the mature subset being dominant in the lung and the immature subset being dominant in the liver. Several experimental approaches, including adoptive transfer, clearly indicated that the immature hepatic NK cell subset, CD27+ CD11b–, was protective against liver metastasis; this subset mediated its protection by a perforin-dependent cytotoxic mechanism. In contrast, the more mature NK cell subsets were more efficient at reducing pulmonary tumor load. These data indicate that organ-specific immune responses may play a pivotal role in determining the permissiveness of a given organ for the establishment of a metastatic niche.     

Expression of renal cell protein markers is dependent on initial mechanical culture conditions.

The rotating wall vessel is optimized for suspension culture, with laminar flow and adequate nutrient delivery, but minimal shear. However, higher shears may occur in vivo. During rotating wall vessel cultivation of human renal cells, size and density of glass-coated microcarrier beads were changed to modulate initial shear. Renal-specific proteins were assayed after 2 days. Flow cytometry antibody binding analysis of vitamin D receptor demonstrated peak expression at intermediate shears, with 30% reduction outside this range. Activity of cathepsin C showed the inverse pattern, lowest at midshear, with twofold increases at either extreme. Dipeptidyl-peptidase IV had no shear dependence, suggesting that the other results are specific, not universal, changes in membrane trafficking or protein synthesis. On addition of dextran, which changes medium density and viscosity but not shear, vitamin D receptor assay showed no differences from controls. Neither cell cycle, apoptosis/necrosis indexes, nor lactate dehydrogenase release varied between experiments, confirming that the changes are primary, not secondary to cell cycling or membrane damage. This study provides direct evidence that mechanical culture conditions modulate protein expression in suspension culture.     

The association of human coagulation factors VIII,IXa and X with phospholipid vesicles involves both electrostatic and hydrophobic interactions

Blood coagulation factor X (FX) is converted to its active form (FXa) by a membrane bound multi-protein enzyme complex, comprised of factor VIII (FVIII), factor IXa (FIXa) and FX. Characterization of the molecular forces involved in the association of these proteins with phospholipids is crucial to understanding how these proteins bind to the lipid milieux of physiological membranes. In this report, the molecular forces involved in the association of FVIII, FIXa or FX with phospholipid vesicles (PLV) were characterized by ligand affinity chromatographic analyses. Treating FVIII-affinity columns with agents that disrupt electrostatic interactions caused elution of 15.2% of the total bound PLV, while agents that disrupt hydrophobic interactions caused elution of 84.8% of the total bound PLV. These results demonstrate that the association of PLV with FVIII is primarily hydrophobic. In contrast, the association of PLV with FIXa or FX is largely the result of electrostatic forces. This was established by observing that 71.3% and 78.9% of the total bound PLV was eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt electrostatic interactions. Of the total bound PLV, 28.7% and 21.2% were eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt hydrophobic interactions. These data demonstrate that hydrophobic forces play a heretofore unrecognized role in the association of PLV with FIXa or FX.     

Metabolic cost of generating muscular force in human walking: insights from load-carrying and speed experiments.

We sought to understand how leg muscle function determines the metabolic cost of walking. We first indirectly assessed the metabolic cost of swinging the legs and then examined the cost of generating muscular force during the stance phase. Four men and four women walked at 0.5, 1.0, 1.5, and 2.0 m/s carrying loads equal to 0, 10, 20, and 30% body mass positioned symmetrically about the waist. The net metabolic rate increased in nearly direct proportion to the external mechanical power during moderate-speed (0.5-1.5 m/s) load carrying, suggesting that the cost of swinging the legs is relatively small. The active muscle volume required to generate force on the ground and the rate of generating this force accounted for >85% of the increase in net metabolic rate across moderate speeds and most loading conditions. Although these factors explained less of the increase in metabolic rate between 1.5 and 2.0 m/s ( approximately 50%), the cost of generating force per unit volume of active muscle [i.e., the cost coefficient (k)] was similar across all conditions [k = 0.11 +/- 0.03 (SD) J/cm3]. These data indicate that, regardless of the work muscles do, the metabolic cost of walking can be largely explained by the cost of generating muscular force during the stance phase.