Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Asymptomatic Bacteriuria Escherichia coli Are Live Biotherapeutics for UTI

Urinary tract infections (UTI) account for approximately 8 million clinic visits annually with symptoms that include acute pelvic pain, dysuria, and irritative voiding. Empiric UTI management with antimicrobials is complicated by increasing antimicrobial resistance among uropathogens, but live biotherapeutics products (LBPs), such as asymptomatic bacteriuria (ASB) strains of E. coli, offer the potential to circumvent antimicrobial resistance. Here we evaluated ASB E. coli as LBPs, relative to ciprofloxacin, for efficacy against infection and visceral pain in a murine UTI model. Visceral pain was quantified as tactile allodynia of the pelvic region in response to mechanical stimulation with von Frey filaments. Whereas ciprofloxacin promoted clearance of uropathogenic E. coli (UPEC), it did not reduce pelvic tactile allodynia, a measure of visceral pain. In contrast, ASB E. coli administered intravesically or intravaginally provided comparable reduction of allodynia similar to intravesical lidocaine. Moreover, ASB E. coli were similarly effective against UTI allodynia induced by Proteus mirabilis, Enterococccus faecalis and Klebsiella pneumoniae. Therefore, ASB E. coli have anti-infective activity comparable to the current standard of care yet also provide superior analgesia. These studies suggest that ASB E. coli represent novel LBPs for UTI symptoms.     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

The 3T3 cells were treated with 50 mu g/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughout the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[3H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase.     

Adenylate cyclase activity of NIH 3T3 cells morphologically transformed by ras genes

The observed homology between G-proteins which regulate adenylate cyclase and ras proteins and the suggested role of ras in the regulation of adenylate cyclase in yeast prompted us to examine the regulation of adenylate cyclase in three cell lines: (i) NIH 3T3 cells, (ii) NIH 3T3 cells transformed by high levels of the normal rasH gene product and (iii) NIH 3T3 cells transformed by a mutated rasH gene product. We found that the regulation of adenylate cyclase by G-proteins is identical in the three cell lines, although the response of the transformed NIH 3T3 cells to agonists is strongly attenuated. Our data suggest that mammalian ras products do not interact directly with adenylate cyclase, although their increased expression may indirectly inhibit the interaction of adenylate cyclase stimulatory receptors with G-proteins.     

Observatons on the growth and metabolic functions of cultured cells derived from human adipose tissue.

The growth and metabolic activity of cultured cells derived from human adipose tissue (CAT cells) were studied and compared to cultured skin fibroblasts. The morphological appearance of the CAT cells was distinctly different from that of fibroblasts. The growth rate of CAT cells as measured by 3H-thymidine incorporation was much slower than the fibroblast growth rate. Cultured CAT cells synthesized significantly 14C-glucose, while fibroblast cultures had a higher metabolic rate as measured by CO2 production. Insulin stimulated 3H-thymidine incorporation in both CAT and fibroblast cultures. The CAT cells did not show a consistent insulin response of lipid or CO2 production, but this may be a reflection of donor age or nutritional status. Even though the CAT cell may be a type of stromal cell peculiar to adipose tissue rather than a preadipocyte or adipocyte, it may prove useful in studies of human obesity.     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

Summary The 3T3 cells were treated with 50 μg/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughouth the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[³H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase. This work was supported by institutional funds granted by Texas Woman's University.